Methods of increasing satellite cell proliferation with vorinostat or bosutinib

ABSTRACT

The invention provides methods for inducing, enhancing or increasing satellite cell proliferation, and an assay for screening for a candidate compound for inducing, enhancing or increasing satellite cell proliferation. Also provided are methods for repairing or regenerating a damaged muscle tissue of a subject.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/728,476, filed Oct. 9, 2017, which is a continuation of U.S.application Ser. No. 15/012,656, filed Feb. 1, 2016, which is acontinuation-in-part of, and claims priority to, co-pending U.S. patentapplication Ser. No. 14/126,716, filed Jun. 13, 2014, which is anational stage filing under 35 U.S.C. 371 of International ApplicationNo. PCT/US2012/042964, filed Jun. 18, 2012, which claims benefit under35 U.S.C. § 119(e) of U.S. Provisional Application No. 61/497,708, filedJun. 16, 2011, the contents of which are incorporated herein byreference in their entirety.

GOVERNMENT SUPPORT

This invention was made with government support under HL127365 awardedby the National Institutes of Health. The government has certain rightsin the invention.

TECHNICAL FIELD

The present disclosure relates generally to compositions and methods ofpromoting satellite cell proliferation.

BACKGROUND

Satellite cells are a population of skeletal muscle stem cells that arelocated beneath the basal lamina surrounding the muscle fiber and arerequired for muscle growth and muscle repair after injury or exercise.Satellite cell number and function are affected by normal aging and inseveral diseases, resulting in progressive muscle wasting or inefficientrecovery after injury. Examples include Duchenne muscular dystrophy, inwhich satellite cells are depleted through constant use, and sarcopenia,where satellite cells may both be depleted and adversely affected intheir proliferative capacity by changes in their environment (Jejurikarand Kuzon, Apoptosis, 8 (2003), 573-578). Finding treatments that wouldexpand the endogenous population of satellite cells could aid greatly intreating these debilitating diseases.

Thus, there is need in the art for compositions and methods for inducingsatellite cell proliferation.

SUMMARY

The inventors conducted an image-based screen of selected smallmolecules for their ability to increase proliferation in satellite cellsisolated from adult mouse muscle tissue. Satellite cells were isolatedusing cell surface markers (Sherwood et al., Cell, 119 (2004), 543-554),cultured in the presence of small molecules for four days, and thenanalyzed using automated confocal microscopy to determine cell number.Using this procedure, the inventors discovered several compounds,operating through defined signaling pathways, which can enhanceproliferation in cultures of primary mouse satellite cells. The myogeniccapacity of treated cells is currently being tested through markeranalysis and differentiation assays, as well as in vivo muscle grafting.The compounds are capable of proliferating cells without adverselyaffecting their differentiation potential and can be used for treatmentof diseases having a skeletal muscle defect as one of their components.These compounds are also referred to as proliferation enhancers herein.

Accordingly, presented herein is a method of inducing, enhancing orincreasing satellite cell proliferation. The method comprisingcontacting a satellite cell with a compound selected from the groupconsisting of kinase inhibitors, G protein coupled receptor (GPCR)modulators, histone deacetylases (HDAC) modulators, hedgehog signalingpathway modulators, neuropeptides, dopamine receptor modulators,serotonin receptor modulators, histamine receptor modulators, adenosinereceptor agonists, ionophores, ion channel modulators, gamma-secretasemodulators, corticosteroids, and any combinations thereof. The satellitecell to be contacted can be in vitro, ex vivo or in vivo.

In another aspect, provided herein is a method for repairing orregenerating a damaged muscle tissue of a subject. The method comprisingadministering to the subject a therapeutically effective amount of acompound selected from the group consisting of kinase inhibitors, Gprotein coupled receptor (GPCR) modulators, histone deacetylases (HDAC)modulators, hedgehog signaling pathway modulators, neuropeptides,dopamine receptor modulators, serotonin receptor modulators, histaminereceptor modulators, ionophores, ion channel modulators, gamma-secretasemodulators, and any combinations thereof.

In yet another aspect, provided herein is a method of screening for acandidate compound for inducing, enhancing or increasing satellite cellproliferation. The method comprising: (a) contacting a population ofsatellite cells with a test compound; (b) assessing satelliteproliferation; and (c) selecting the compound that induces, increases orenhances satellite cell proliferation.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawings will be provided by the Office upon request and paymentof the necessary fee.

FIGS. 1A-1C show Opera analysis of satellite cell number. Images werecaptured using the Perkin Elmer Opera automated confocal imaging system.Since all cells isolated from the CAG-EGFP animals were fluorescent, theinventors could image the cells directly (FIG. 1A). Images were analyzedusing Acapella software to count cells that had signals within the setthresholds for each parameter (FIG. 1B). Cells or debris that were toodim (arrow), or too small (arrowheads) were excluded. Cells and debristhat were highly fluorescent or too large in size were also excluded.Cells could then be further grouped according to set criteria, such asroundness (FIG. 1C).

FIGS. 2A-2D show analysis of positive hit compounds. Adult mousesatellite cells were isolated via FACS, based on the procedure outlinedin Sherwood et al. (Cell, 119 (2004), 543-554). Then, cells were treatedwith compounds and allowed to proliferate for four days, fixed, andimaged. Proliferation induced by compounds was compared to that foundwith a DMSO vehicle control (FIG. 2A), or to that found with bFGF (FIG.2B). Two example images from positive hit compounds are shown, Flt3Kinase inhibitor (FIG. 2C) and Adenosine receptor agonist (FIG. 2D).

FIGS. 3A and 3B show validation of some exemplary hit compounds.Compounds identified in the primary screen as hits were then tested in adose response assay. The level of proliferation is measured as foldchange over the DMSO vehicle control value of (A.1±0.3 (FIG. 3A) and1±0.4 (FIG. 3B). For comparison, the bFGF positive control values were3.4±0.9 (FIG. 3A) and 5.6±1.7 (FIG. 3B).

FIG. 4 shows optimization of culture conditions. Culture conditions wereoptimized by testing the effects of compounds with differential exposuretime, with or without the presence of bFGF. For all time points, mediawere replaced with standard proliferation medium on the indicated days.For this compound, the inclusion of bFGF had an additive effect onproliferation. Also, while the compound itself led to proliferationsimilar to the bFGF positive control at all time points, it was seen tobe more effective with a shorter exposure time.

FIGS. 5A-5C show differentiation of treated cultures. CAG-EGFP satellitecells were grown in the inventors' standard proliferation conditions andexposed to compounds for four days. Under the inventors' standardproliferation conditions cells were cultured on laminin-coated plates inHam's F-10 medium supplemented with 10% heat inactivated horse serum,100 Units/mL penicillin/100 ug/mL streptomycin, and 2 mM L-glutamine.Compounds were added the day after plating. The compounds and media wereeither refreshed daily or after three days. To differentiate cells andform myotubes, after four days in proliferation conditions, media wasswitched to high-glucose DMEM supplemented with 10% heat inactivatedhorse serum, 10% fetal bovine serum, 0.5% chick embryo extract, 100Units/mL penicillin/100 ug/mL streptomycin, and 2 mM L-glutamine.Cultures were grown in differentiation media for 3-5 days until myotubeformation was observed, then fixed. Cultures were then switched intodifferentiation media and cultured another four days. They were thenfixed and stained with anti-myosin heavy chain (red channel) and Hoechst(blue channel). Cells grown with only DMSO vehicle control did not formmyotubes because they were not dense enough in culture (data not shown).Cells grown in the presence of bFGF positive control (FIG. 5A) were ableto form large myotubes. Cultures grown in the presence of Flt3 Kinaseinhibitor (FIG. 5B) and adenosine agonist (FIG. 5C) were able to formmyotubes as well.

FIG. 6 shows myogenic colony formation of single SMPs treated withCEP/DMSO for 5 days. Treatment interval d1-d3.5 and n=3.

FIG. 7 shows Exposure to compound from d2 to d4.5. Initial cell numberwas 250 and n=3.

FIGS. 8A and 8B show expression of CXCR4 and Beta-1 integrin on culturedSMPs after 5 days: CEP (FIG. 8A) and DMSO (FIG. 8B).

FIGS. 9A-13 show dose response curves for some exemplary hit compounds.Shown are Sunitinib (SU11248, FIGS. 9A and 9B), Jak3 inhibitor VI (FIGS.10A and 10B), Lestaurtinib (CEP701, FIGS. 11A and 11B), Bosutinib (FIG.12), and SU11652 (FIG. 13).

FIGS. 14-17 are bar graphs showing the synergistic effect of bFGF withCEP701 (FIGS. 14 and 15) and Jak3 inhibitor VI (FIG. 17), and Sunitinib(FIG. 17) on proliferation of satellite cells.

FIG. 18-20 are photographs showing differentiation of satellite cellsafter treatment with CEP701 (FIG. 18), Sunitinib (FIG. 19), and Jak3inhibitor VI (FIG. 20).

FIG. 21 is a bar graph showing the synergistic effect of CEP701 with aTGF-beta inhibitor (Alk5 inhibitor II).

FIG. 22 shows specificity of CEP701 for proliferating satellite cells.Left panel: CEP701; right panel: DMSO.

FIG. 23 is bar graph showing CEP701 has no effect on primary fibroblasts

FIGS. 24 and 25 are bar graphs showing CEP701 is effective in both theaged (FIG. 24) and young (FIG. 25) tissue.

FIGS. 26 and 27 are line graph showing dose response curves forN6-cyclopentyladenosine (FIG. 26) and Budesonide (FIG. 27).

FIGS. 28 and 29 are bar graph showing the synergistic effect of bFGFwith N6-cyclopentyladenosine (FIG. 28) and Budesonide (FIG. 29).

FIGS. 30 and 31 are photographs showing differentiation of satellitecells after treatment with N6-Cyclopentyladenosine (FIG. 30) andBudesonide (FIG. 31).

FIG. 32 is a bar graph showing the synergistic effect ofN6-Cyclopentyladenosine with a TGF-beta inhibitor (Alk5 inhibitor II).

FIG. 33 illustrates the screening assay performed to identify thoseprimary and secondary compounds that were shown to increase satellitecell proliferation in vitro.

FIG. 34 depicts the customer screening library used by the presentinventors to screen a set of approximately 400 compounds to identifythose compounds that increase satellite cell proliferation.

FIG. 35 illustrates the results of assays performed and that demonstratethat lestaurtinib (CEP701), Sunitinib (SU11248), JAK3 inhibitor VI, andN6-cyclopentyladenosine (CPA) were found to increase in vitro satellitecell proliferation. Lestaurtinib (CEP701) was identified as a top hit,was effective at nanomolar doses and had target overlap with severalother hit compounds.

FIG. 36 illustrates the results of an assay performed and that evidencethat lestaurtinib (CEP701) increased proliferation of aged satellitecells in vitro.

FIGS. 37A-37B illustrate a dose response assay (FIG. 37A) and theresponse curves for each of lestaurtinib (CEP701), sunitinib (SU11248),JAK3 inhibitor VI, and N6-cyclopentyladenosine (CPA) (FIG. 37B).

FIG. 38 demonstrates that both CEP-701 and AC220 increased humansatellite cells by more than 2-fold at a concentration of 1 nM relativeto control (DMSO).

FIG. 39 presents the results of an assay performed and confirms thatthose compounds identified as hits (e.g., CEP701, SU11248, JAK3inhibitor VI, CPA and Tyr AG490) drive myoblast differentiation.

FIG. 40 demonstrates that CEP701 enhances myoblast differentiation indifferentiation media relative to the DMSO control, as evidenced by theobserved increase in both myoblast area and length.

FIGS. 41A-41B depict an experimental protocol (FIG. 41A) and the resultsof that experiment (FIG. 41B) and which illustrates that CEP701treatment of cells resulted in an increased number of GFP+ fibers persection, relative to control (DMSO).

FIGS. 42A-42D depict an experimental protocol (FIG. 42A) and thecorresponding results observed. As illustrated in FIGS. 42B-42D,treatment with CEP701 increased both regenerating fiber size andsatellite cell number in vivo in both adult and aged mice.

FIGS. 43A-43B illustrate the affect that the identified compounds haveon RTKs (FIG. 43A) and, as illustrated in FIG. 43B, compares the foldchange in phospho-RET in both uninjured contralateral tibialis anterior(TA) muscle to that observed 2 days post-cardiotoxin injury. Asillustrated in FIG. 43B, 2 days post-cardiotoxin injury, anapproximately 8-fold increase in phospho-RET was observed by ELISArelative to uninjured contralateral TA muscle.

FIGS. 44A-44B illustrate that satellite cells express RET in vitro.

FIGS. 45A-45D illustrate that CEP701 treatment inhibits RETphosphorylation in vitro.

FIG. 46 shows the results of a study evaluating the effects of the invitro deletion of RET.

FIG. 47 illustrates that by using a conditional RET mutant and reporter,the present inventors were able to determine that the RET promoter isactive in at least 25% of satellite cells.

FIG. 48 shows that that RET knockout cells proliferate better thanwild-type cells in vitro (n=6; p=0.0004).

FIG. 49 demonstrates the fold change relative to control of untreatedFLT3 and RET knockout cells.

DETAILED DESCRIPTION OF EXEMPLARY EMBODIMENTS

Provided herein is a method of increasing satellite cell proliferation.The method comprising contacting a satellite cell with a compoundselected from the group consisting of kinase inhibitors, G proteincoupled receptor (GPCR) modulators, histone deacetylase (HDAC)modulators, hedgehog signaling pathway modulators, neuropeptides,dopamine receptor modulators, serotonin receptor modulators, histaminereceptor modulators, adenosine receptor agonists, ionophores, ionchannel modulators, adenosine receptor modulators, gamma-secretasemodulators, corticosteroids, any combination thereof.

As used herein, the term “proliferation” means growth and division ofcells. In some embodiments, the term “proliferation” as used herein inreference to cells refers to a group of cells that can increase innumber over a period of time.

As used herein, “inducing,”, “enhancing,” or “increasing” satellite cellproliferation means that satellite cells replicate at a faster rateand/or more frequently. In some embodiments of this and other aspectsdescribed herein, satellite cell proliferation is increased by at least5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold,1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold ormore higher relative to an untreated control. The % or fold increase insatellite cell proliferation can be determined by measuring number ofreplicating satellite cells while in contact with a compound describedherein relative to a control where the satellite cells are not incontact with the compound. Increase in proliferation can also be basedon ratios of replicating cells to total number of cells in therespective treated and untreated control. In some embodiments, totalnumber of cells in the treated and untreated controls is used todetermine the proliferation. Satellite cell proliferation can bedetermined using the BrdU incorporation method described in U.S. PatentPublication No. 2009/0136481, content of which is incorporated herein byreference.

Myosatellite cells or satellite cells are small mononuclear progenitorcells with virtually no cytoplasm found in mature muscle. They are foundsandwiched between the basement membrane and sarcolemma (cell membrane)of individual muscle fibers, and can be difficult to distinguish fromthe sub-sarcolemmal nuclei of the fibers. Satellite cells are able todifferentiate and fuse to augment existing muscle fibers and to form newfibers. These cells represent the oldest known adult stem cell niche,and are involved in the normal growth of muscle, as well as regenerationfollowing injury or disease.

In undamaged muscle, the majority of satellite cells are quiescent; theyneither differentiate nor undergo cell division. In response tomechanical strain, satellite cells become activated. Activated satellitecells initially proliferate as skeletal myoblasts before undergoingmyogenic differentiation.

Markers characteristic of satellite cells include the expression of cellsurface proteins or the encoding genes, the expression of intracellularproteins or the encoding genes, cell morphological characteristics, andthe like. Those skilled in the art will recognize that knownimmunofluorescent, immunochemical, polymerase chain reaction, in situhybridization, Northern blot analysis, chemical or radiochemical orbiological methods can readily ascertain the presence or absence ofsatellite cell specific characteristics.

If desired, the type(s) of cells in a population of satellite can bedetermined using techniques that are well known in the art. For example,the use of cell-type specific stains. Alternatively, one can performimmunofluorescence staining using antibodies directed to varioussatellite cell specific proteins. In addition, a cell type can bedetermined by its morphology using techniques such as, for example,light microscopy, or electron microscopy.

Satellite cells express a number of distinctive genetic markers. Forexample, current thinking is that all satellite cells express PAX7 andPAX3 (F. Rlaix et al. Nature, 2005, 435(7044): 898-899). Activatedsatellite cells express myogenic transcription factors, such as Myf5 andMyoD. They also begin expressing muscle-specific filament proteins suchas desmin as they differentiate.

Little is known of the regulation of satellite cells. Whilst togetherPAX3 and PAX7 currently form the definitive satellite markers, Pax genescan be poor transcriptional activators. The dynamics of activation andquiesence and the induction of the myogenic program through the myogenicregulatory factors, Myf5, MyoD, myogenin, and MRF4 remains to bedetermined. There is some research indicating that satellite cells arenegatively regulated by a protein called myostatin. Increased levels ofmyostatin up-regulate a cyclin-dependent kinase inhibitor called p21 andthereby induce the differentiation of satellite cells.

In some embodiments, the satellite cells are in a stabilized state,e.g., the cells were taken from a subject and treated in such a manneras to allow them to be stored for some period of time. For example, thecells can be frozen, e.g., using methods known in the art for freezingprimary cells, such that the cells are viable when thawed. For example,methods known in the art to freeze and thaw embryos to generate livemammals can be adapted for use in the present methods. Such methods caninclude the use of liquid nitrogen, e.g., with one or morecryoprotectants, e.g., agents that prevent freeze-thaw damage to thecell.

Kinase Inhibitors

As used herein, the term “kinase” means any phosphotransferase enzymethat transfers a phosphate group. In some embodiments, the kinase is aprotein kinase. Protein kinases are a family of enzymes that catalysethe phosphorylation of specific residues in proteins. In general proteinkinases fall into several groups; those which preferentiallyphosphorylate serine and/or threonine residues, those whichpreferentially phosphorylate tyrosine residues and those whichphosphorylate both tyrosine and Ser/Thr residues.

Protein kinases include, for example, but are not limited to, members ofthe Protein Tyrosine Kinase family (PTKs), which in turn can be dividedinto the cytoplasmic PTKs and the receptor PTKs (RTKs). The cytoplasmicPTKS include the SRC family, (including: BLK; FOR; FYN; HCK; LCK; LYN;SRC; YES and YRK); the BRK Family (including: BRK; FRK, SAD; and SRM);the CSK family (including: CSK and CTK); the BTK family, (including:BTK; ITK; TEC; MKK2 and TXK), the Janus kinase family, (including: JAKI,JAK2, JAK3 and Tyk2), the FAK family (including, FAK and PYK2); the Fesfamily (including FES and FER), the ZAP70 family (including ZAP70 andSYK); the ACK family (including ACK1 and ACK2); and the Abl family(including ABL and ARG). The RTK family includes the EGF-Receptor family(including, EGFR, HER2, HER3 and HER4); the Insulin Receptor family(including INS-R and IGF1-R); the PDGF-Receptor family (includingPDGFRa, PDGFR|3, CSF1R, KIT, FLK2); the VEGF-Receptor family (including;FLT1, FLK1 and FLT4); the FGF-Receptor family (including FGFR1, FGFR2,FGFR3 and FGFR4); the CCK4 family (including CCK4); the MET family(including MET and RON); the TRK family (including TRKA, TRKB, andTRKC); the AXL family (including AXL, MER, and SKY); the TIE/TEK family(including TIE and TIE2/TEK); the EPH family (including EPHA1, EPHA2,EPHA3, EPHA4, EPHA5, EPHA6, EPHA7, EPHA8, EPHB1, EPHB2, EPHB3, EPHB4,EPHB5, EPHB6); the RYK family (including RYK); the MCK family (includingMCK and TYRO 10); the ROS family (including ROS); the RET family(including RET); the LTK family (including LTK and ALK); the ROR family(including ROR1 and ROR2); The Musk family (including Musk); the LMRfamily including LMR1, LMR2 and LMR3); and the SuRTK106 family(including SuRTK106).

Representative, non-limiting examples of kinases include Abl,Abl(T3151), ALK, ALK4, AMPK, Arg, Arg, ARKS, ASK1, Aurora-A, Axl, Blk,Bmx, BRK, BrSK1, BrSK2, BTK, CaMKI, CaMKII, CaMKIV, CDKl/cyclinB,CDK2/cyclinA, CDK2/cyclinE, CDK3/cyclinE, CDK5/p25, CDK5/p35,CDK6/cyclinD3, CDK7/cyclinH/MATl, CDK9/cyclin Tl, CHK1, CHK2, CKl(y),CK18, CK2, CK2a2, cKit(D816V), cKit, c-RAF, CSK, cSRC, DAPK1, DAPK2,DDR2, DMPK, DRAK1, DYRK2, EGFR, EGFR(L858R), EGFR(L861Q), EphA1, EphA2,EphA3, EphA4, EphAS, EphAV, EphAS, EphB1, EphB2, EphB3, EphB4, ErbB4,Per, Fes, FGFR1, FGFR2, FGFR3, FGFR4, Fgr, Fill, Flt3(D835Y), Flt3,Flt4, Fms, Fyn, GSK3(3, GSK3a, Hck, HIPK1, HIPK2, HIPK3, IGF-1R, IKK(3,IKKa, IR, IRAKI, IRAK4, IRR, ITK, JAK2, JAK3, JNKIal, JNK2a2, JNK3, KDR,Lck, LIMK1, LKB1, LOK, Lyn, Lyn, MAPK1, MAPK2, MAPK2, MAPKAP-K2,MAPKAP-K3, MARK1, MEK1, MELK, Met, MINK, MKK4, MKK6, MKK7(3, MLCK, MLK1,Mnk2, MRCK-beta, MRCKa, MSK1, MSK2, MSSK1, MST1, MST2, MST3, MuSK, NEK2,NEKS, NEK6, NEK7, NLK, p70S6K, PAK2, PAK3, PAK4, PAK6, PAR-lBa, PDGFR(3,PDGFRa, PDK1, PI3K beta, PI3K delta, PI3K gamma, Pim-1, Pim-2, PKA(b),PKA, PKB(3, PKBa, PKBy, PKC{circumflex over ( )}i, PKC(3I, PKC(3II,PKCa, PKCy, PKC8, PKCe, PKC{circumflex over ( )}, PKCr|, PKC9, PKCi,PKD2, PKG1(3, PKGla, Plk3, PRAK, PRK2, PrKX, PTK5, Pyk2, Ret, RIPK2,ROCK-I, ROCKII, ROCK-II, Ron, Ros, Rse, Rskl, Rskl, Rsk2, Rsk3, SAPK2a,SAPK2a(T106M), SAPK2b, SAPK3, SAPK4, SGK, SGK2, SGK3, SIK, Snk, SRPK1,SRPK2, STK33, Syk, TAKI, TBK1, Tie2, TrkA, TrkB, TSSK1, TSSK2, WNK2,WNK3, Yes, ZAP-70, ZIPK. In some embodiments, the kinases may be ALK,Aurora-A, Axl, CDK9/cyclin Tl, DAPK1, DAPK2, Per, FGFR4, GSK3(3, GSK3a,Hck, JNK2a2, MSK2, p70S6K, PAK3, PI3K delta, PI3K gamma, PKA, PKB(3,PKBa, Rse, Rsk2, Syk, TrkA, and TSSK1. In yet other embodiments thekinase is selected from the group consisting of ABL, AKT, AURORA, CDK,DBF2/20, EGFR, EPH/ELK/ECK, ERK/MAPKFGFR, GSK3, IKKB, INSR, JAK DOM 1/2,MARK/PRKAA, MEK/STE7, MEKK/STE11, MLK, mTOR, PAK/STE20, PDGFR, PI3K,PKC, POLO, SRC, TEC/ATK, and ZAP/SYK.

Similarly, the serine/threonine specific kinases comprise a number ofdistinct sub-families, including; the extracellular signal regulatedkinases, (p42/ERK2 and p44/ERKI); c-Jun NH2-terminal kinase (JNK);cAMP-responsive element-binding protein kinases (CREBK); cAMP dependentkinase (CAPK); mitogen-activated protein kinase-activated protein kinase(MAPK and its relatives); stress-activated protein kinase-p38/SAPK2;mitogen-and stress-activated kinase (MSK); protein kinases, PKA, PKB andPKC inter alia.

In some embodiments, the kinase is a FMS-like tyrosine kinase 3 (Flt3),PDGFR/EGFR, Bcr-abl, Jak3, or SRC kinase inhibitor. Flt3 is also knownas FLK2 (Fetal Liver Kinase-2) and STK1 (human Stem Cell Kinase-1).

As used herein, the term “kinase inhibitor” means any compound, moleculeor composition that inhibits or reduces the activity, e.g.,phosphotransferase activity, of a kinase. Without limitations, a kinaseinhibitor can be selected from the group consisting of small or largeorganic or inorganic molecules; monosaccharides; disaccharides;trisaccharides; oligosaccharides; polysaccharides; biologicalmacromolecules, e.g., proteins, peptides, peptide analogs andderivatives thereof, peptidomimetics, nucleic acids, nucleic acidanalogs and derivatives, enzymes, antibodies, portion or fragments ofantibodies; an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues; naturally occurring orsynthetic compositions; and any combinations thereof.

A wide variety of kinase inhibitors are known in the art and can be usedin the compositions and methods described herein. A kinase inhibitor canbe selected form the group consisting of FMS-like tyrosine kinase 3inhibitor; Aurora kinase inhibitor; Aurora-B kinase inhibitor; Aurora-Ckinase inhibitor; Beta-adrenergic receptor kinase inhibitor; Check pointkinase inhibitor; Cyclin-dependent kinase 1 inhibitor; Cyclin-dependentkinase 2 inhibitor; Cyclin-dependent kinase 4 inhibitor;Cyclin-dependent kinase inhibitor; EphB2 kinase inhibitor; Epidermalgrowth factor receptor kinase inhibitor; N-acylmannosamine kinaseinhibitor; MAP kinase inhibitor; Opheline kinase inhibitor;Phosphatidylinositol 3-kinase beta inhibitor; Phosphatidylinositol3-kinase gamma inhibitor; Protein kinase (CK1) inhibitor; Protein kinaseB inhibitor; Protein kinase C eta inhibitor; Protein-serine-threoninekinase inhibitor; Proto-oncogene tyrosine-protein kinase Fyn inhibitor;Proto-oncogene tyrosine-protein kinase Kit inhibitor; Pyridoxal kinaseinhibitor; Raf kinase B inhibitor; Raf kinase inhibitor; Rho-associatedkinase inhibitor; Ribosomal protein S6 kinase inhibitor; and anycombination thereof.

In certain aspects, the compounds disclosed herein are RET kinaseinhibitors. In certain aspects, the compounds disclosed herein (e.g.,CEP-701 and/or AC220) are or comprise a RET inhibitor or otherwiseinhibit RET phosphorylation. In certain aspects, the compounds disclosedherein (e.g., CEP-701 and/or AC220) are or comprise a C-RET inhibitor orotherwise inhibit C-RET phosphorylation.

Also contemplated are compounds and compositions that reduce RET kinaseligands. For example, in certain aspects the compounds and compositionsdisclosed comprise an antibody or agent that interferes with RET kinaseactivation, such as antibodies and agents that bind to or otherwiseinterfere with the binding of a C-RET ligand (e.g., GDNF) to C-RET.

Kinase inhibitors amenable to the compositions and methods describedherein are also described, for example in U.S. Pat. Nos. 5,674,998;5,795,977; 5,864,033; 6,194,939; 6,239,133; 6,346,625; 6,391,894;6,448,277; 6,492,409; 6,498,165; 6,706,711; 6,723,726; 6,825,190;6,825,355; 6,943,161; 6,951,859; 6,982,266; 6,982,266; 7,056,925;7,101,884; 7,105,531; 7,105,531; 7,115,597; 7,153,856; 7,183,307;7,196,090; 7,199,137; 7,199,147; 7,223,757; 7,232,826; 7,262,199;7,265,134; 7,309,787; 7,314,940; 7,326,713; 7,326,713; 7,449,488;7,456,169; 7,459,554; 7,470,693; 7,470,713; 7,488,826; 7,504,429;7,511,040; 7,514,435; 7,517,882; 7,521,460; 7,528,132; 7,550,478;7,550,598; 7,572,914; 7,582,652; 7,598,272; 7,601,852; 7,618,982;7,635,703; 7,648,987; 7,662,977; 7,683,060; 7,687,506; 7,732,613;7,749,994; 7,767,674; 7,790,739; 7,812,166; 7,820,662; 7,855,211;7,872,031; 7,893,064; 7,893,081; 7,901,894; 7,915,443; 7,943,629;7,968,546; 7,994,159; 7,998,507; 8,022,057; 8,024,821; 8,026,234;8,026,246; 8,026,247; 8,044,221; 8,093,239; 8,093,383; 8,143,410;8,148,361; and 8,152,630 and U.S. Patent Publication Nos. 20070161673;20090181940; 20090215785; 20100097654; 20100234404; 20110008211;20030044203; 20030065180; 20030087919; 20030119839; 20030139462;20030187001; 20030199511; 20030199525; 20030216446; 20040034038;20040034075; 20040082581; 20040180897; 20040192725; 20050043347;20050096324; 20050131022; 20050153990; 20050171076; 20050187247;20050192304; 20050203114; 20050215556; 20050239794; 20050239815;20050261318; 20050267133; 20050277642; 20050277642; 20050288290;20050288321; 20050288321; 20060019958; 20060058304; 20060058341;20060079563; 20060122389; 20060122389; 20060148824; 20060178388;20060217369; 20060264438; 20060270694; 20060276490; 20060281789;20060287370; 20060287381; 20070049600; 20070054906; 20070060619;20070078140; 20070099856; 20070099935; 20070123534; 20070173516;20070173525; 20070185139; 20070191420; 20070191420; 20070203143;20070213386; 20070254896; 20070259869; 20070270425; 20070280928;20080027063; 20080108611; 20080153869; 20080161297; 20080167330;20080207613; 20080207613; 20080207632; 20080255155; 20080255184;20080269244; 20080293714; 20080293785; 20080312307; 20090054425;20090054436; 20090105209; 20090124602; 20090131407; 20090131437;20090131506; 20090149389; 20090162376; 20090175852; 20090197862;20090215750; 20090221616; 20090233960; 20090264446; 20090286779;20090298855; 20090318440; 20100004234; 20100041645; 20100041684;20100041684; 20100048599; 20100081662; 20100093767; 20100099710;20100113454; 20100120772; 20100120801; 20100144732; 20100144745;20100160303; 20100168102; 20100179134; 20100179146; 20100190816;20100204221; 20100222342; 20100234386; 20100298301; 20100317643;20100324041; 20100331314; 20110009410; 20110070317; 20110077237;20110118285; 20110124623; 20110136789; 20110190280; 20110195980;20110257238; 20110269739; 20110269772; 20110275630; 20110281857;20110281866; 20110288097; 20110293745; 20110294812; 20120015937;20120041024; 20120053187; 20120065213; 20120071490; 20120071494;20120077851; 20120095014; 20120095233; 20120212961; 20100267774; and20100324074, content of all of which is incorporated herein byreference.

In some embodiments, the kinase inhibitor can be selected from the groupconsisting of

and any combination thereof.

In some embodiments, the kinase inhibitor is or comprises quizartinib(AC220), or any salt, ester or chelate thereof. In certain embodiments,the kinase inhibitor is

In some embodiments of this and other aspects described herein, activityof the kinase is inhibited or lowered by at least 5%, at least 10%, atleast 20%, at least 30%, at least 40%, at least 50%, at least 60%, atleast 70%, at least 80%, at least 90%, at least 95%, at least 98%, or100% (e.g. complete loss of activity) relative to an uninhibitedcontrol. Without wishing to be bound by theory, activity of a kinase canbe determined using any assay known in the art for measuring theactivity of the kinase, e.g., by measuring phosphorylation reactions.

Hedgehog Signaling Pathway Modulators

As used herein, the term “modulate,” with reference to the Hedgehogsignaling pathway, means to regulate positively or negatively the normalfunctioning of a component in the Hedgehog signaling pathway. Thus, theterm modulate can be used to refer to an increase, decrease, masking,altering, overriding or restoring the normal functioning of a componentin the Hedgehog signaling pathway.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the hedgehog signaling pathway by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, or at least 95%, at least 98% or more relativeto a control with no modulation.

The term “Hedgehog signaling pathway”, “Hedgehog pathway” and “Hedgehogsignal transduction pathway” are all used to refer to the chain ofevents normally mediated by Hedgehog, smoothened, Ptch1, and Gli, amongothers, and resulting in a changes in gene expression and otherphenotypic changes typical of Hedgehog activity. Activating a downstreamcomponent can activate the Hedgehog pathway even in the absence of aHedgehog protein. For example, overexpression of smoothened willactivate the pathway in the absence of Hedgehog, Gli and Ptch1 geneexpression are indicators of an active Hedgehog-signaling pathway.Accordingly, compounds described herein can be used to overcome aninappropriate increase in Hedgehog signal transduction, whether saidincrease in signal transduction is the result in a mutation/lesion in acomponent of the Hedgehog signaling pathway (e.g., Ptch1, Gli1, Gli3,smoothened, etc.) or whether said increase in signal transduction occursin the context of a cell which does not comprise a mutation/lesion in acomponent of the Hedgehog signaling pathway (e.g., a wildtype cell withrespect to components of the Hedgehog signaling pathway). Thus, in someembodiments, the cell has a phenotype of smoothened gain-of-function,Hedgehog gain-of-function, patched (Ptc) loss-of-function, Gligain-of-function, and/or over expression of Hedgehog ligands.

The term “smoothened gain-of-function” refers to an aberrantmodification or mutation of a smo gene, or an increased level ofexpression of the gene, which results in a phenotype that resemblescontacting a cell with a Hedgehog protein, e.g., aberrant activation ofa Hedgehog pathway. While not wishing to be bound by any particulartheory, it is noted that Ptch1 may not signal directly into the cell,but rather modulates the activity of smoothened, another membrane boundprotein located downstream of Ptch1 in Hedgehog signaling (Marigo etal., (1996) Nature 384: 177-179; Taipale et al. (2002) Nature 418,892-896). The gene smo is a segment polarity gene required for thecorrect patterning of every segment in Drosophila (Alcedo et al., (1996)Cell 86:221232). Human homologs of smo have been identified. See, forexample, Stone et al. (1996) Nature 384:129-134, and GenBank accessionU84401. The smoothened gene encodes an integral membrane protein withcharacteristics of heterotrimeric G-protein-coupled receptors; i.e.,7-transmembrane regions. This protein shows homology to the DrosophilaFrizzled (Fz) protein, a member of the wingless pathway. Ptc is a Hhreceptor. Cells that express Smo fail to bind Hh, indicating that smodoes not interact directly with Hh (Nusse, (1996) Nature 384: 119120).Rather, the binding of Sonic Hedgehog (SHH) to its receptor, PTCH isthought to prevent normal inhibition by PTCH of smoothened. Activatingsmoothened mutations are known to occur in sporadic basal cell carcinoma(Xie, et al., Nature, 1998, 391: 90-92), and in primitiveneuroectodermal tumors of the central nervous system (Reifenberger, etal., Cancer Res., 1998, 58:1798-1803).

The term “Hedgehog gain-of-function” refers to an aberrant modificationor mutation of a Ptch1 gene, Hedgehog gene, or smoothened gene, or adecrease (or loss) in the level of expression of such a gene, whichresults in a phenotype which resembles contacting a cell with a Hedgehogprotein, e.g., aberrant activation of a Hedgehog pathway. Thegain-of-function may include a loss of the ability of the Ptch1 geneproduct to regulate the level of expression of Ci homolog genes, e.g.,Gli 1, Gli2, and Gli3. The term “Hedgehog gain-of-function” is also usedherein to refer to any similar cellular phenotype (e.g., exhibitingexcess proliferation) that occurs due to an alteration anywhere in theHedgehog signal transduction pathway, including, but not limited to, amodification or mutation of Hedgehog itself. For example, a tumor cellwith an abnormally high proliferation rate due to activation of theHedgehog signaling pathway would have a “Hedgehog gain-of-function”phenotype, even if Hedgehog is not mutated in that cell.

The term “patched loss-of-function” refers to an aberrant modificationor mutation of a Ptch1 gene, or a decreased level of expression of thegene, which results in a phenotype which resembles contacting a cellwith a Hedgehog protein, e.g., aberrant activation of a Hedgehogpathway. The loss-of-function may include a loss of the ability of thePtch1 gene product to regulate the level of expression or activity of Cihomolog genes, e.g., Gli1, Gli2, and Gli3. The term ‘Ptch1 loss-offunction’ is also used herein to refer to any similar cellular phenotype(e.g., exhibiting excess proliferation) that occurs due to an alterationanywhere in the Hedgehog signal transduction pathway, including, but notlimited to, a modification or mutation of Ptch1 itself. For example, atumor cell with an abnormally high proliferation rate due to activationof the Hedgehog signaling pathway would have a “Ptch1 loss-of-function”phenotype, even if Ptch1 is not mutated in that cell.

The term “Gli gain-of-function” refers to an aberrant modification ormutation of a Gli gene, or an increased level of expression of the gene,which results in a phenotype that resembles a cell responding to aHedgehog protein, e.g., aberrant activation of a Hedgehog pathway.

The vertebrate family of Hedgehog genes includes three members thatexist in mammals, known as Desert (Dhh), Sonic (Shh) and Indian (Ihh)Hedgehogs, all of which encode secreted proteins. These various Hedgehogproteins consist of a signal peptide, a highly conserved N-terminalregion, and a more divergent C-terminal domain. Biochemical studies haveshown that autoproteolytic cleavage of the Hh precursor protein proceedsthrough an internal thioester intermediate which subsequently is cleavedin a nucleophilic substitution. It is likely that the nucleophile is asmall lipophilic molecule which becomes covalently bound to theC-terminal end of the N-peptide, tethering it to the cell surface. Thebiological implications are profound. As a result of the tethering, ahigh local concentration of N-terminal Hedgehog peptide is generated onthe surface of the Hedgehog producing cells. It is this N-terminalpeptide which is both necessary and sufficient for short- and long-rangeHedgehog signaling activities.

An inactive Hedgehog signaling pathway is where the transmembraneprotein receptor Patched (Ptc) inhibits the activity of Smoothened(Smo), a seven transmembrane protein. The transcription factor Gli, adownstream component of Hh signaling, is processed to a repressor formand nuclear accumulation of activator forms prevented throughinteractions with cytoplasmic proteins, including Fused and Suppressorof fused (Sufu). As a consequence, transcriptional activation ofHedgehog target genes is repressed. Activation of the pathway isinitiated through binding of any of the three mammalian ligands (Dhh,Shh or Ihh) to Ptc. Ligand binding results in a reversal of therepression of Smo, thereby activating a cascade that leads to thetranslocation of the active form of the transcription factor Gli to thenucleus. Nuclear Gli activates target gene expression, including Ptc andGli itself. Increased levels of Hedgehog signaling are sufficient toinitiate cancer formation and are required for tumor survival.

The Hedgehog signaling pathway modulator can be an agonist or antagonistof the hedgehog signaling pathway.

The term “hedgehog agonist” refers to an agent which antagonizes orblocks the bioactivity of patched, such as to increase transcription oftarget genes. The hedgehog antagonists can be used to overcome a ptcgain-of-function and/or a smoothened loss-of-function, the latter alsobeing referred to as “smoothened agonists” The term “hedgehogantagonist” likewise refers not only to any agent that may act bydirectly inhibiting the normal function of the hedgehog protein, butalso to any agent that inhibits the hedgehog signaling pathway, and thusrecapitulates the function of ptc.

Exemplary hedgehog signaling pathway modulators include, but are notlimited to, AY9944, triparanol, jervine, cyclopamine, tomatidine, andthe like.

GPCR Modulators

As used herein, the term “modulate,” with reference to the GPCRs, meansto regulate positively or negatively the normal functioning of a GPCRsignaling pathway. Thus, the term modulate can be used to refer to anincrease, decrease, masking, altering, overriding or restoring thenormal functioning of a GPCR. A GPCR modulator can be a GPCR agonist ora GPCR antagonist.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the GPCR by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 95%, at least 98% or more relative to a control with nomodulation.

The G protein-coupled receptors (GPCRs) form a vast superfamily of cellsurface receptors which are characterized by an amino-terminalextracellular domain, a carboxyl-terminal intracellular domain, and aserpentine structure that passes through the cell membrane seven times.Hence, such receptors are sometimes also referred to as seventransmembrane (7TM) receptors. These seven transmembrane domains definethree extracellular loops and three intracellular loops, in addition tothe amino- and carboxy-terminal domains. The extracellular portions ofthe receptor have a role in recognizing and binding one or moreextracellular binding partners (e.g., ligands), whereas theintracellular portions have a role in recognizing and communicating withdownstream molecules in the signal transduction cascade.

In all, GPCRs can be grouped into 6 classes based on sequence homologyand functional similarity: Class A (or 1) (Rhodopsin-like); Class B (or2) (Secretin receptor family); Class C (or 3) (Metabotropicglutamate/pheromone); Class D (or 4) (Fungal mating pheromonereceptors); Class E (or 5) (Cyclic AMP receptors); and Class F (or 6)(Frizzled/Smoothened). The very large rhodopsin A group has been furthersubdivided into 19 subgroups (A1-A19). More recently, an alternativeclassification system called GRAFS (Glutamate, Rhodopsin, Adhesion,Frizzled/Taste2, Secretin) has been proposed.

As used herein, the term “GPCR ligand” refers to molecules that bindGPCRs. The G protein-coupled receptors bind a variety of ligandsincluding calcium ions, hormones, chemokines, neuropeptides,neurotransmitters, nucleotides, lipids, odorants, and even photons, andare important in the normal (and sometimes the aberrant) function ofmany cell types. [See generally Strosberg, Eur. J. Biochem. 196:1-10(1991) and Bohm et al, Biochem J. 322:1-18 (1997).] When a specificligand binds to its corresponding receptor, the ligand typicallystimulates the receptor to activate a specific heterotrimericguanine-nucleotide-binding regulatory protein (G-protein) that iscoupled to the intracellular portion of the receptor. The G protein inturn transmits a signal to an effector molecule within the cell, byeither stimulating or inhibiting the activity of that effector molecule.These effector molecules include adenylate cyclase, phospholipases andion channels. Adenylate cyclase and phospholipases are enzymes that areinvolved in the production of the second messenger molecules cAMP,inositol triphosphate and diacyglycerol. It is through this sequence ofevents that an extracellular ligand stimuli exerts intracellular changesthrough a G protein-coupled receptor. Each such receptor has its owncharacteristic primary structure, expression pattern, ligand-bindingprofile, and intracellular effector system.

GPCRs include receptors for sensory signal mediators (e.g., light andolfactory stimulatory molecules); adenosine, bombesin, bradykinin,endothelin, γ-aminobutyric acid (GABA), hepatocyte growth factor (HGF),melanocortins, neuropeptide Y, opioid peptides, opsins, somatostatin,GH, tachykinins, members of the vasoactive intestinal peptide family,and vasopressin; biogenic amines (e.g., dopamine, epinephrine,norepinephrine, histamine, glutamate (metabotropic effect), glucagon,acetylcholine (muscarinic effect), and serotonin); chemokines; lipidmediators of inflammation (e.g., prostaglandins, prostanoids,platelet-activating factor, and leukotrienes); and peptide hormones(e.g., calcitonin, C5a anaphylatoxin, follicle-stimulating hormone(FSH), gonadotropin-releasing hormone (GnRH), neurokinin,thyrotropin-releasing hormone (TRH), cannabinoids, and oxytocin). GPCRsthat act as receptors for stimuli that have not yet been identified areknown as orphan receptors.

Whereas, in other types of receptors that have been studied, whereinligands bind externally to the membrane, the ligands of GPCRs typicallybind within the transmembrane domain. However, protease-activatedreceptors are activated by cleavage of part of their extracellulardomain.

Types of GPCR ligands include, but are not limited to: agonists whichshift the equilibrium in favor of active states; inverse agonists whichshift the equilibrium in favor of inactive states; and neutralantagonists which do not affect the equilibrium. When a GPCR in anactive state encounters a G-protein, it can activate the G-protein.GPCRs are the target of about 40% of all prescription pharmaceuticals onthe market. (Filmore, Modern Drug Discovery, November 2004, pp. 11).Examples of commonly prescribed GPCR-based drugs include Atenolol(TENORMIN®), Albuterol (VENTOLIN®), Ranitidine (ZANTAC®), Loratadine(CLARITIN®), Hydrocodone (VICODIN®) Theophylline (THEODUR®), andFluoxetine (PROZAC®).

Exemplary GPCR modulators include, but are not limited to, corticotropinreleasing factor (CRF), urocortin 1, urocortin 2, usorcortin 3,parathyroid hormone, PTH-related hormone, TIP39, calcitonin, amylin,CGRP (CALCA and CALCB), adrenomedullin, secretin, VIP, PACAP, glucagon,GHRH, GLP-1, GLP-2, Dynorphin A, Dynorphin A amide, Dynorphin A (1-6),Dynorphin A (1-13), Dynorphin A (2-13), Dynorphin A (2-17), MetEnk,Met-Enk-RF-amide, Met-Enk-Arg-Phe, Met-Enk-Glyleu, [D-pGlu1, D-Phe2,D-Trp3,6]-LH-RH, g1-MSH amide, g2-MSH, [N-MePhel, D-Pro4]-Morphiceptin(PLO17), ACTH (Human), Leu-Enk, Adrenomedullin (22-52), Adrenomedullin(26-52) (Human)(ADM antagonist), Agouti 1-40 Amide, Agouti RelatedProtein (87-132)-Amide, Alpha-MSH, Alpha-Neo-Endorphin, Amylin Amide,BAM(1-20), BAM(1-22), BAM(2-22), BAM(6-22), BAM(1-20), ANP (AtrialNatriuretic Peptide), Anti-Inflammatory Peptide 1, Anti-InflammatoryPeptide 2, (3-endorphin, Benzylureido-Met-Leu-Phe, Beta-ANP,Beta-Endorphin, Beta-MSH, Big Endothelin-1, Big Gastrin-1, BNP (BrainNatriuretic Peptide-32), BNP-45 (Cardiac Natriuretic Peptide, Bombesin,BAM(8-25), BAM(8-20), FLRF, Calcitonin Gene Related Peptide, NPFF,Calcitonin, Calcitonin Gene Related Peptide (8-37), CART (55-1,02), CART(55102)[Met(O)67, CART (61-102), CGRP (8-37), CGRP II, CholecystokininOctapeptide [CCK(26-33)], Cholecystokinin-33, CNP-22 (C-Type NatriureticPeptide), Corticotropin Releasing Factor, Cortistatin-14, NPAF, SST,NPY, FMRFamide, OrpaninFQFMRF amide related peptide, YMRFamide,YLPLRFamide, YFMRFamide, LPLRFamide, dFMRFamide, W-Nle-R-F-amide, andACEP.

Polypeptide modulators of GPCRs include, but are not limited to,vasopressin, oxytocin, somatostatin, neuropeptide Y, GnRH, leutinizinghormone, follicle stimulating hormone, parathyroid hormone, orexins,urotensin II, endorphins, enkephalins, and the like. A list of GPCRmodulators is compiled on the web atpharminfo.pharm.kyoto-u.ac.jp/services/glida/ligand_classification.php

In some embodiments, the GPCR modulator inhibits binding of a ligand byat least about or about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 100% relative to a control. Binding of a ligand to aGPCR can be determined by any method known to one of skill in the art.

In some embodiments, the GPCR modulator reduces an activity of a GPCR byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100% (e.g. complete loss of activity)relative to an uninhibited control.

In some embodiments, the GPCR modulator enhances an activity of a GPCRby at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold,1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative toan unactivated control.

In some embodiments, the GPCR modulator is capable of binding to theactive site of a GPCR (e.g., a binding site for a ligand).

In some embodiments, the serotonin receptor modulator is capable ofbinding to an allosteric site of a GPCR.

In some embodiments of this and other aspects described herein, the GPCRantagonist has an IC50 of less than or equal to 500 nM, less than orequal to 250 nM, less than or equal to 100 nM, less than or equal to 50nM, less than or equal to 10 nM, less than or equal to 1 nM, less thanor equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equalto 0.001 nM.

In some embodiments of this and other aspects of the invention, the GPCRagonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

In some embodiments the GPCR modulator can be selected from the groupconsisting of naltrindole

methoctramine tetrahydrochloride

and any combination thereof.Dopamine Receptor Modulators

As used herein, the term “dopamine receptor modulator” refers tocompounds that modulate one or more dopamine receptors. A dopaminereceptor modulator can be a dopamine agonist or a dopamine antagonist.As used herein, the term “dopamine agonist” refers to compounds thatactivate and/or stimulate one or more dopamine receptors and/or increaselevels of dopamine (such as L-dopa or drugs which inhibit dopaminemetabolism) and/or stimulate a dopamine signaling pathway and/or reducelevels of norepinephrine, and/or inhibit a norepinephrine signalingpathway. The term “dopamine agonist” also includes analogs of dopaminemolecules which exhibit at least some biological activity in common withnative human dopamine receptors. As such, the term “dopamine agonist”encompasses dopaminergic agents. As used herein the term “dopaminergicagent” refers to compounds which mimic the action of dopamine.Accordingly, the term dopaminergic agent is intended to encompassdopamine, derivatives of dopamine, and compounds which have dopaminelike actions on dopamine receptors. Exemplary analogs of dopamineinclude the ergolines and the aporphines such an apomorphine, pergolide,bromocriptine and lisuride).

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the dopamine receptor by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, at least 98% or more relative to a controlwith no modulation.

Without wishing to be bound by a theory, a dopamine agonist can act viaone of several pathways. For example, a dopamine agonist can activate orpotentiate D1 dopamine receptors and/or Dj-like receptors such as D1 andD5 dopamine receptors and/or D2 dopamine receptors (e.g., D2, D2 shortand D2 long receptors, D4, and D4 dopamine receptors) and/or D3 dopaminereceptors and/or D4 dopamine receptors. A dopamine agonist can act byinhibiting one or more enzyme involved in biosynthesis and/ortransformation and/or breakdown of dopamine.

Exemplary dopamine agonists include, but are not limited to,(−)-7-{[2-(4-Phenylpiperazin-1-yl)ethyl]propylamino}-5,6,7,8-tetrahydronaphthalen-2-ol;(+)-4-propyl-9-hydroxynaphthoxazine ((+)PHNO);(E)-1-aryl-3-(4-pyridinepiperazin-1-yl)propanone oximes;(R)-3-(4-Propylmorpholin-2-yl)phenol (PF-219,061); (R,R)—S32504;2-(N-phenylethyl-N-propylamino)-5-hydroxytetralin;2-bromo-a-ergocriptine (bromocriptine);5,6,7,8-Tetrahydro-6-(2-propen-1-yl)-4H-thiazolo[4,5-d]azepin-2-amine(BHT-920); 5-HT uptake inhibitor; 5-HT-1A agonists (such as roxindole);6-Br-APB; 6-methyl-8-a-(N-acyl)amino-9-ergoline;6-methyl-8-a-(N-phenyl-acety)amino-9-ergoline;6-methyl-8R-carbobenzyloxy-aminoethyl-10-a-ergoline;7,8-Dihydroxy-5-phenyl-octahydrobenzo[h]isoquinoline;8-acylaminoergoline; 9,10-dihydroergocomine; a2-adrenergic antagonist(such as terguride); A-412,997; A-68,930; A-77,636; A-86,929; ABT-670;ABT-724; AF-14; alaptide; amisulpride; anyD-2-halo-6-alkyl-8-substituted ergoline; Aplindore; Apomorphine;Aripiprazole (Abilify in USA); benzazepine analogs; BP-897;Bromocriptine; bromocriptine mesylate; Cabergoline;cis-8-Hydroxy-3-(n-propyl)-1,2,3a,4,5,9b-hexahydro-1H- andtrans-N-{4-[4-(2,3-Dichlorophenyl)-1-piperazinyl]cyclohexyl}-3-methoxybenzamide;clozapine; COMT inhibitors (such as CGP-28014, entacapone andtolcapone); CP-226,269; CP-96,345; CY-208,243;D-2-bromo-6-methyl-8-cyanomethylergoline; Dihydrexidine;dihydro-alpha-ergocriptine; dihydro-alpha-ergotoxine;dihydroergocriptine; dihydroergocryptine; dihydroergotoxine (hydergine);Dinapsoline; Dinoxyline; domperidone; Dopamine; dopamine D1 receptoragonists; dopamine D2 receptor agonists; dopamine D3 receptor agonists;dopamine D4 receptor agonists; dopamine D5 receptor agonists; dopamineuptake inhibitors (such as GBR-12909, GBR-13069, GYKI-52895, andNS-2141); doprexin; Doxanthrine; ER-230; erfotoxine; Ergocomine;ergoline derivatives; ergot alkaloid derivatives; eticlopride;etisulergine; FAUC 299; FAUC 316; Fenoldopam; Flibanserin; haloperidol;iloperidone; L-dopa; levodopa; Lisuride; lisuride; LSD; LU111995;mazapertine; Methylphenidate; monoamine oxidase-B inhibitors (such asselegiline, N-(2butyl)-N-methylpropargylamine, N-methyl-N-(2-pentyl)propargylamine, AGN-1133, ergot derivatives, lazabemide, LU-53439,MD-280040 and mofegiline); N-0434; Naxagolide; olanzapine; opiatereceptor agonists (such as NIH-10494); PD-118,440; PD-168,077; Pergolide(such as A-68939, A-77636, dihydrexine, and SKF-38393); PIP3EA;piribedil; Piribedil; Pramipexole; Quinagolide; Quinelorane; Quinpirole;racemic trans-10,11-dihydroxy 5,6,6a, 7,8,12b-hexahydro and relatedbenzazepine analogs; raclopride; remoxipride; risperidone; Ro10-5824;Ropinirole; Rotigotine; Salvinorin A; SDZ-HDC-912; sertindole;SKF-38,393; SKF-75,670; SKF-81,297; SKF-82,526 (fenoldopam); SKF-82,598;SKF-82,957; SKF-82,958; SKF-38,393; SKF-77,434; SKF-81,297; SKF-82,958;SKF-89,145; SKF-89,626; spiperone; spiroperidol; sulpride; sumanirole;Talipexole; Terguride; tropapride; WAY-100635; YM 09151-2; zetidoline;β-adrenergic receptor agonists; and analogs, derivatives, enantiomers,metabolites, prodrugs, and pharmaceutically acceptable salts thereof.

Exemplary beta-3 adrenergic receptor agonists include, but are notlimited to, DPDMS; dopexamine; AJ-9677; AZ-40140; BMS187413; BMS-194449;BMS-210285; BRL-26830A; BRL-28410; BRL-35135; BRL-37344; CGP 12177;CL-316243; CP-114271; CP-331648; CP-331679; D-7114; FR-149175; GW-2696;GW-427353; ICI-198157; L-750355; L-796568; LY-377604; N-5984; SB-226552;SR-58611A; SR-59062A; SWR0342SA; ZD-2079; and analogs, derivatives,enantiomers, metabolites, prodrugs, and pharmaceutically acceptablesalts thereof.

In some embodiments, the dopamine agonist enhances an activity of adopamine receptor by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%,80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold ormore relative to an unactivated control.

In some embodiments, the dopamine agonist inhibits the binding of aligand to its receptor by at least about or about any one of 10%, 20%,30%, 40%, 50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control.

In some embodiments, the dopamine receptor modulator is capable ofbinding to the active site of a dopamine receptor (e.g., a binding sitefor a ligand).

In some embodiments, the dopamine receptor modulator is capable ofbinding to an allosteric site of a dopamine receptor.

In some embodiments of this and other aspects described herein, thedopamine agonist has an IC50 of less than or equal to 500 nM, less thanor equal to 250 nM, less than or equal to 100 nM, less than or equal to50 nM, less than or equal to 10 nM, less than or equal to 1 nM, lessthan or equal to 0.1 nM, less than or equal to 0.01 nM, or less than orequal to 0.001 nM.

In some embodiments of this and other aspects of the invention, thedopamine agonist has an EC50 of less than or equal to 500 nM, 250 nM,100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

In some embodiments, the dopamine agonist inhibits the dopaminebeta-hydroxylase. Dopamine beta-hydroxylase converts dopamine tonorepinephrine. Thus, by inhibiting dopamine beta-hydroxylase,intracellular dopamine is increased while norepinephrine is decreased.

Exemplary inhibitors of DBH include, but are not limited to fusaricacid; 1,1′,1″,1′″-[disulfanediylbis-(carbonothioylnitrilo)]tetraethane(disulflram); 2-Hydroxy-2,4,6-cycloheptatrien-1-one (tropolone, alsoreferred to as 2-Hydroxytropone or Purpurocatechol);5-(aminomethyl)-1-[(2S)-5,7-difluoro-1,2,3,4-tetrahydronaphthalen-2-yl]-1,3-dihydro-2H-imidazole-2-thione(Nepicastat, INN, or SYN117)); 1-(4-hydroxybenzyl)imidazole-2-thiol;FLA-63; diethyidithiocarbamate; betachlorophenethylamine;4-hydroxybenzyl cyanide; 2-halo-3(p-hydroxyphenyl)-1-propene;1-phenyl-1-propyne; 2-phenylallylamine; 2-(2-thienyl)allylamine;2-thiophene-2(2-thienyl)allylamine; 3-phenylpropargylamine; 1-phenyl-1(aminoethyl)ethane; N-(trifluoroacetyl)phenyl(aminoethyl) ethane;5-picolinic acid substituted with an alkyl group containing up to 6carbon atoms; 5-picolinic acid substituted with a halo alkyl groupcontaining up to 6 carbon atoms; and analogs, derivatives, enantiomers,metabolites, prodrugs, and pharmaceutically acceptable salts thereof.

Other inhibitors of dopamine beta-hydroxylase include, but are notlimited to U.S. Pat. Nos. 4,487,761; 4,634,711; 4,719,223; 4,743,613;4,749,717; 4,761,415; 4,762,850; 4,798,843; 4,810,800; 4,835,154;4,839,371; 4,859,779; 4,876,266; 4,882,348; 4,906,668; 4,935,438;4,963,568; 4,992,459; 5,100,912; 5,189,052; 5,597,832; 6,407,137;6,559,186; 7,125,904; 7,576,081, content of all of which is hereinincorporated by reference in their entirety.

In some embodiments of this and other aspects of the invention, activityof the dopamine beta-hydroxylase is inhibited or lowered by at least 5%,at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, atleast 60%, at least 70%, at least 80%, at least 90%, at least 95%, atleast 98%, or 100% (e.g. complete loss of activity) relative to anuninhibited control.

In some embodiments, the dopamine beta-hydroxylase inhibitor has thedesired activity at a concentration that is lower than the concentrationof the inhibitor that is required to produce another, unrelatedbiological effect. In some exemplary embodiments, the concentration ofthe inhibitor required for dopamine beta-hydroxylase inhibitory activityis at least about 2-fold lower, or at least about 5-fold lower, or atleast about 10-fold lower, or at least about 20-fold lower than theconcentration required to produce an unrelated biological effect.

In some embodiments of this and other aspects described herein, thedopamine beta-hydroxylase inhibitor has an IC50 of less than or equal to500 nM, less than or equal to 250 nM, less than or equal to 100 nM, lessthan or equal to 50 nM, less than or equal to 10 nM, less than or equalto 1 nM, less than or equal to 0.1 nM, less than or equal to 0.01 nM, orless than or equal to 0.001 nM.

In some embodiments, the dopamine receptor modulator inhibits binding ofa ligand by at least about or about any one of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% relative to a control. Binding of aligand to a dopamine receptor can be determined by any method known toone of skill in the art.

In some embodiments, the dopamine receptor modulator reduces an activityof a dopamine receptor by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g.complete loss of activity) relative to an uninhibited control.

In some embodiments, the dopamine receptor modulator enhances anactivity of a dopamine receptor by at least 5%, 10%, 20%, 30%, 40%, 50%,50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold or more relative to an unactivated control.

In some embodiments, the dopamine receptor modulator is capable ofbinding to the active site of a dopamine receptor (e.g., a binding sitefor a ligand).

In some embodiments, the serotonin receptor modulator is capable ofbinding to an allosteric site of a dopamine receptor.

In some embodiments of this and other aspects described herein, the GPCRantagonist has an IC50 of less than or equal to 500 nM, less than orequal to 250 nM, less than or equal to 100 nM, less than or equal to 50nM, less than or equal to 10 nM, less than or equal to 1 nM, less thanor equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equalto 0.001 nM.

In some embodiments of this and other aspects of the invention, the GPCRagonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

Serotonin Receptor Modulators

As used herein, the term “modulate,” with reference to the serotoninreceptors means to regulate positively or negatively the normalfunctioning of the serotonin receptor. Thus, the term modulate can beused to refer to an increase, decrease, masking, altering, overriding orrestoring the normal functioning of a serotonin receptor. A serotoninreceptor modulator can be an agonist or an antagonist of the serotoninreceptor.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the serotonin receptor by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 95%, at least 98% or more relative to acontrol with no modulation.

Serotonin (5-hydroxytryptamine, 5-HT) is a major neurotransmittereliciting effects via a multiplicity of receptors. To date, at leastfifteen different 5-HT receptors have been identified, largely as theresult of cloning cDNA's, and these receptors have been grouped intoseven families (5-HT₁ through 5-HT₇). See, for example, Hoyer, et al.,Pharmacol. Biochem. Behav. 2002, 71: 533-554. Fourteen of the fifteencloned 5-HT receptors are expressed in the brain. 5-HT is implicated inmany disease states, particularly conditions of the central nervoussystem including; depression, anxiety, schizophrenia, eating disorders,obsessive compulsive disorder, learning and memory dysfunction,migraine, chronic pain, sensory perception, motor activity, temperatureregulation, nociception, sexual behavior, hormone secretion, andcognition.

As used herein, the term “serotonin receptor modulator” intends andencompasses a compound that binds to or inhibits binding of a ligand toa serotonin receptor or reduces or eliminates or increases or enhancesor mimics an activity of a serotonin receptor. As such, a “serotoninreceptor modulator” encompasses both a serotonin receptor antagonist anda serotonin receptor agonist. In some embodiments, the serotoninreceptor modulator binds to or inhibits binding of a ligand to a 5-HT1Aand/or a 5-HT1B and/or a 5-HT2A and/or a 5-HT2B and/or a 5-HT2C and/or a5-HT3 and/or a 5-HT4 and/or a 5-HT6 and/or a 5-HT7 receptor or reducesor eliminates or increases or enhances or mimics an activity of a 5-HT1Aand/or a 5-HT1B and/or a 5-HT2 A and/or a 5-HT2B and/or a 5-HT2C and/ora 5-HT3 and/or a 5-HT4 and/or a 5-HT6 and/or a 5-HT7 receptor in areversible or irreversible manner.

In some embodiments, the serotonin modulator is a serotonin receptorantagonist.

Exemplary serotonin modulators include, but are not limited to, (−)cisapride; (−) norcisapride; (−) venlafaxine; (+) cisapride; (+)norcisapride; (+) venlafaxine;1-(2-fluorophenyl)-3-(4-hy)-prop-2-en-1-one-O-(2-dimethylaminoethyl)-oxime;[5[3-(4-methylsulphonylamino)-benzy-1-1,2,4-oxadiazol-5yl]-1H-indol-3-yl]ethanamine(L694247); 2-hydroxymethylolanzapine; 2-Methyl-5-HT; 2C-B;3-tropanyl-indole-3-carboxylate; 3-tropanyl-indole-3-carboxylatemethiodide; 311C90; 5-CT; 5-MeO-DMT; 5-MT; 8-OH-DPAT; A-372,159;Agomelatine; AL-38022A; Almotriptan; alnitidan; alosetron; Alosetron;alpha-Me-5-HT; Alprenolol; Amitriptyline; AR-A000002; Aripiprazole;AS-19; Asenapine; BIMU-8; BMY 7378; BRL-15572; Bufotenin; Buspirone;BVT-933 (Biovitrum); BW-723C86; BZP; Cannabidiol; chlorpromazine;cilansetron; Cinitapride; Cisapride; citalopram; Clomipramine;Clozapine; enanserin; CP-93,129; CP-94,253; Cyanopindolol; Dazopride;demethylcitalopram; demethylsertraline; desipramine;desmethylolanzapine; Dihydroergotamine; Dimebolin; DMT; Dolasetron; DOM;EGIS-12233; Eletriptan; Eletriptan; EMD-386,088; EMDT; Eplivanserin;Etoperidone; Fenfluramine; Flesinoxan; Flibanserin; Fluoxetine;fluphenazine; fluvoxamine; Frovatriptan; Gepirone; GR 127935;Granisetron; haloperidol; homochlorcyclizine; hydrodolasetron;Iloperidone; Imipramine; lodocyanopindolol; Ipsapirone; Ketanserin;1-[5(2-thienylmethoxy)-1H-3-indolyl[propan-2-amine hydrochloride(BW723C86); L-Lysine; Lecozotan; Lisuride; Lorcaserin; loxapine; LSD;LY-278,584; LY-53,857; m-chlorophenylpiperazine (MCPP); MDL 11939; MDMA;Mefway; Memantine; Mescaline; Metergoline; Methiothepin; methiothepin;Methysergide; Metoclopramide; Mianserin; Mirtazapine; Mosapride; MS-245;Myristicin; NAN-190; Naratriptan; Naratriptan; Nefazodone; norcisapride;Norfenfluramine; norfluoxetine; nortriptaline; Olanzapine; Ondansetron;oxetorone; Oxprenolol; p-NPPL; paroxetine; perlapine; Piboserod;Pimavanserin; Pindolol; piperazine; Pizotifen; Propanolol; Prucalopride;Psilocin; Psilocybin; Quetiapine; Quetiapine; Risperidone; Quipazine;R-hydroxynefazodone; r(−) fluoxetine; r(+) ondansetron; Rauwolscine;Renzapride; renzapride; risatriptan; Risperidone; Ritanserin;Rizatriptan; Ro04-6790; Robalzotan; RS-56812; RS-67333; RU 24969; RU24969; s(+) fluoxetine; S15535; SB 206553; SB 216641; SB 242084;SB-258,585; SB-269,970; SB-271,046; SB-357,134; SB-399,885; SB-699,551;SDZ-205,557; sertraline; sibutramine; Spiperone; Sumatriptan;Tandospiroe; Tegaserod; TFMPP; Trazodone; Tropisetron; Tryptamine;UH-301; Urapidil; Valerenic Acid; venlafaxine; WAY-100,135; WAY-100,635;Xaliproden; YM-348; Yohimbine; Zacopride; zalospirone; zatosetron;Ziprasidone; Zolmitriptan; and analogs, derivatives, enantiomers,prodrugs and pharmaceutically acceptable salts thereof.

Additional serotonin modulators include the compounds described in U.S.Pat. Nos. 4,737,496; 4,782,063; 4,788,290; 4,789,673; 4,797,406;4,903,691; 5,001,133; 5,017,582; 5,130,313; 5,143,916; 5,202,318;5,232,924; 5,260,303; 5,319,085; 5,356,934; 5,399,557; 5,434,161;5,516,782; 5,591,749; 5,604,239; 5,612,366; 5,705,509; 5,728,835;5,736,544; 5,874,429; 5,962,448; 6,187,772; 6,255,306; 6,235,745;6,271,223; 6,288,101; 6,316,468; 6,353,008; 6,436,964; 6,638,934;6,686,374; 6,743,913; 6,828,330; 6,911,452; 7,109,339; 7,244,722;7,297,711; 7,351,707; 7,375,114; 7,592,355; 7,655,691; 7,772,239;7,781,476; and 7,851,474, and U.S. Pat. App. Pub. No. 2003/0153576; No.2005/0215555; No. 2006/0003990; No. 2006/0025601; No. 2006/0079567; No.2006/0100266; No. 2006/0178366; No. 2007/0032481; No. 2007/0244086; No.2010/0004264; and No. 2010/0069356, content of all of which isincorporated herein by reference in their entirety.

In some embodiments, the serotonin receptor modulator inhibits bindingof a ligand by at least about or about any one of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control. Binding of aligand to a serotonin receptor can be determined by an assay described,for example, in U.S. Pat. App. Pub. No. 2009/0239854, content of whichis incorporated herein by reference in its entirety.

In some embodiments, the serotonin receptor modulator reduces anactivity of a serotonin receptor by at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%(e.g. complete loss of activity) relative to an uninhibited control.

In some embodiments, the serotonin receptor modulator enhances anactivity of a serotonin receptor by at least 5%, 10%, 20%, 30%, 40%,50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold or more relative to an unactivated control.

In some embodiments, the serotonin receptor modulator is capable ofbinding to the active site of a serotonin receptor (e.g., a binding sitefor a ligand).

In some embodiments, the serotonin receptor modulator is capable ofbinding to an allosteric site of a serotonin receptor.

In some embodiments of this and other aspects of the invention, theserotonin antagonist has an IC50 of less than or equal to 500 nM, lessthan or equal to 250 nM, less than or equal to 100 nM, less than orequal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM,less than or equal to 0.1 nM, less than or equal to 0.01 nM, or lessthan or equal to 0.001 nM.

In some embodiments of this and other aspects of the invention, theserotonin agonist has an EC50 of less than or equal to 500 nM, 250 nM,100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

Histamine Receptor Modulators

As used herein, the term “modulate,” with reference to the histaminereceptors means to regulate positively or negatively the normalfunctioning of the histamine receptor. Thus, the term modulate can beused to refer to an increase, decrease, masking, altering, overriding orrestoring the normal functioning of a histamine receptor. A histaminereceptor modulator can be an agonist or an antagonist of the histaminereceptor.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the histamine receptor by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, or at least 95%, at least 98% or more relative to acontrol with no modulation.

Histamine receptors belong to the superfamily of G protein-coupled seventransmembrane proteins with histamine as their endogenous ligand. Gprotein coupled receptors constitute one of the major signaltransduction systems in eukaryotic cells. Coding sequences for thesereceptors, in those regions believed to contribute to theagonist-antagonist binding site, are strongly conserved across mammalianspecies. Histamine receptors are found in most peripheral tissue andwithin the central nervous system. There are four known histaminereceptors, H₁, H₂, H₃ and H₄.

As used herein, the term “histamine receptor modulator” intends andencompasses a compound that binds to or inhibits binding of a ligand toa histamine receptor or reduces or eliminates or increases or enhancesor mimics an activity of a histamine receptor. As such, a “histaminereceptor modulator” encompasses both a histamine receptor antagonist anda histamine receptor agonist. In some embodiments, the histaminereceptor modulator binds to or inhibits binding of a ligand to ahistamine H1 and/or H2 and/or H3 and/or H4 receptor or reduces oreliminates or increases or enhances or mimics an activity of a histamineH1 and/or H2 and/or H3 and/or H4 receptor in a reversible orirreversible manner.

In some embodiments, the histamine modulator is a histamine receptorantagonist. Exemplary histamine modulators include, but are not limited,A-349,821; ABT-239; Acrivastine; Alimemazine (trimeprazine); Antazoline;Astemizole; Azatadine; Azelastine; Bepotastine; Bilastine; Bisfentidine;BL-6341A; BL-6548; BMY-25271; BMY-25405; BMY-52368; Brompheniramine;Carbinoxamine; Cetirizine; Chlorcyclizine; Chlorphenamine(chlorpheniramine); Chlorpromazine; Cimetidine; Ciproxifan; Clemastine;Clobenpropit; Clozapine; Cyclizine; Cyproheptadine; D-16637; DA-4634;Desloratadine; Dexchlorpheniramine; Dimebon; Dimenhydrinate;Dimetindene; Diphenhydramine; donetidine; Ebastine; ebrotidine;Embramine; etintidine; Famotidine; famotidine; Fexofenadine; FRG-8701;FRG-8813; Haloperidol; HB-408; HE-30-256; Hydroxyzine; ICI-162846;ICIA-5165; impromidine; JNJ 7777120; Ketotifen; L-643728; Lafutidine;lamtidine; Levocabastine; Levocetirizine; Loratadine; loxtidine;lupitidine; Meclozine; Mepyramine; mifentidine; Mizolastine; Nizatidine;nizatidine; Olonzapin; Olopatadine; ORF-17578; Pheniramine; pifatidine;Promethazine; Quetiapine; Quifenadine; ramixotidine; Ranitidine;Ritanserin; roxatidine; SKF-94482; SR-58042; sufotidine; Terfenadine;Thioperamide; tiotidine; VUF-6002; Wy-45727; zaltidine; and analogs,derivatives, enantiomers, prodrugs and pharmaceutically acceptable saltsthereof.

Additional histamine modulators include the compounds described in U.S.Pat. Nos. 3,932,644; 3,980,781; 4,060,621; 4,112,234; 4,117,131;4,145,546; 4,153,793; 4,154,834; 4,159,329; 4,159,329; 4,218,452;4,227,000; 4,234,588; 4,250,316; 4,255,248; 4,307,104; 4,309,433;4,309,433; 4,318,913; 4,337,256; 4,338,328; 4,374,248; 4,374,248;4,375,341; 4,380,639; 4,385,058; 4,385,058; 4,399,294; 4,432,983;4,439,437; 4,442,110; 4,447,611; 4,481,199; 4,485,104; 4,496,567;4,507,296; 4,520,025; 4,521,418; 4,522,943; 4,524,071; 4,526,973;4,529,723; 4,543,352; 4,551,466; 4,608,380; 4,638,001; 4,645,110;4,670,487; 4,681,883; 4,694,008; 4,738,969; 4,745,110; 4,764,612;4,777,179; 4,812,451; 4,812,452; 4,952,589; 5,273,984; 5,486,526;5,541,343; 5,639,775; 5,753,671; 6,420,560; 6,552,047; 6,936,627;7,115,600; 7,205,316; 7,256,205, and U.S. Pat. App. Pub. No.2002/0086859; No. 2004/0138234; No. 2005/0070525; No. 2006/0047114; No.2006/0069087; No. 2007/0238771; No. 2008/0015200; No. 2009/0239854; No.2009/0325927; and No. 2010/0022580, content of all which is incorporatedherein by reference in their entirety.

In some embodiments, the histamine receptor modulator inhibits bindingof a ligand by at least about or about any one of 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90%, 95% or 100% relative to a control.

In some embodiments, the histamine receptor modulator reduces anactivity of a histamine receptor by at least 5%, at least 10%, at least20%, at least 30%, at least 40%, at least 50%, at least 60%, at least70%, at least 80%, at least 90%, at least 95%, at least 98%, or 100%(e.g. complete loss of activity) relative to an uninhibited control.

In some embodiments, the histamine receptor modulator enhances anactivity of a histamine receptor by at least 5%, 10%, 20%, 30%, 40%,50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold,4-fold, 5-fold or more relative to an unactivated control.

In some embodiments, the histamine receptor modulator is capable ofbinding to the active site of a histamine receptor (e.g., a binding sitefor a ligand).

In some embodiments, the histamine receptor modulator is capable ofbinding to an allosteric site of a histamine receptor.

In some embodiments of this and other aspects of the invention, thehistamine antagonist has an IC50 of less than or equal to 500 nM, lessthan or equal to 250 nM, less than or equal to 100 nM, less than orequal to 50 nM, less than or equal to 10 nM, less than or equal to 1 nM,less than or equal to 0.1 nM, less than or equal to 0.01 nM, or lessthan or equal to 0.001 nM.

In some embodiments of this and other aspects of the invention, thehistamine agonist has an EC50 of less than or equal to 500 nM, 250 nM,100 nM, 50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

In some embodiments, the histamine receptor modulator can bemethylhistamine dihydrochloride, i.e., histamineR(−)-alpha-methyl-dihydrochloride

HDAC Modulators

As used herein, the term “modulate,” with reference to the HDAC means toregulate positively or negatively the normal functioning of the HDAC.Thus, the term modulate can be used to refer to an increase, decrease,masking, altering, overriding or restoring the normal functioning of aHDAC. A HDAC modulator can be an agonist or an antagonist of theserotonin receptor.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the HDAC by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 95%, at least 98% or more relative to a control with nomodulation.

The HDACs are a family including at least eighteen enzymes, grouped inthree classes (Class I, II and III). Class I HDACs include, but are notlimited to, HDACs 1, 2, 3, and 8. Class I HDACs can be found in thenucleus and are believed to be involved with transcriptional controlrepressors. Class II HDACs include, but are not limited to, HDACs 4, 5,6, 7, and 9 and can be found in both the cytoplasm as well as thenucleus. Class III HDACs are believed to be NAD dependent proteins andinclude, but are not limited to, members of the Sirtuin family ofproteins. Non-limiting examples of sirtuin proteins include SIRT1-7.

The term “HDAC modulator” as used herein refers to a compound that hasthe ability to modulate transcriptional activity.

In some embodiments, the HDAC modulator is a HDAC inhibitor. The term“HDAC inhibitor” as used herein refers to a compound that has theability to inhibit histone deacetylase activity. This therapeutic classis able to block angiogenesis and cell cycling, and promote apoptosisand differentiation. HDAC inhibitors both display targeted anticanceractivity by itself and improve the efficacy of existing agents as wellas other new targeted therapies.

As used herein, the term “selective HDAC inhibitor” refers to an HDACinhibitor that does not significantly interact with all three HDACclasses. As used herein, a “Class I selective HDAC” refers to an HDACinhibitor that interacts with one or more of HDACs 1, 2, 3 or 8, butdoes not significantly interact with the Class II HDACs (i.e., HDACs 4,5, 6, 7 and 9).

A number of compounds with HDAC inhibitory activity are known in the art(see e.g., Marks et al., J. Natl. Cancer Inst. 92; 1210-1216 (2000) andMiller et al, J. Med. Chem, 46(24); 5097-5115 (2003), incorporatedherein by reference) and can used as an HDAC inhibitory agent of thedisclosure. An HDAC inhibitor can be a short-chain fatty acid, such asbutyric acid, phenylbutyrate (PB), 4-phenylbutyrate (4-PBA),pivaloyloxymethyl butyrate (Pivanex, AN-9), isovalerate, valerate,valproate, valproic acid, propionate, butyramide, isobutyramide,phenylacetate, 3-bromopropionate, or tributyrin as non-limitingexamples. Short-chain fatty acid compounds having HDac inhibitoryactivity are described in U.S. Pat. Nos. 4,988,731, 5,212,326,4,913,906, 6,124,495, 6,110,970 6,419,953, 6,110,955, 6,043,389,5,939455, 6,511,678, 6,528,090, 6,528,091, 6,713,086, 6,720,004, U.S.Patent Publication No. 20040087652, Intl. Publication No. WO 02/007722,and in Phiel et al, J Biol Chem, 276(39):36734-41 (2001), Rephaeli etal, Int J Cancer, 116(2):226-35 (2005), Reid et al. Lung Cancer,45(3):381-6 (2004), Gottlicher et al, 2001, EMBO J, 22(13):3411-20(2003), and Vaisburg et al, Bioorg Med Chem Lett, 14(1):283-7 (2004). AnHDac inhibitor can be compound bearing a hydroxyamic acid group, such assuberoylanlide hydroxamic acid (SAHA), trichostatin A (TSA),trichostatin C (TSC), salicylhydroxamic acid, oxamflatin, subericbishydroxamic acid (SBHA), m-carboxycinnamic acid bishydroxamic acid(CBHA), pyroxamide (CAS RN 382180-17-8), diethylbis-(pentamethylene-N,Ndimethylcarboxamide)malonate (EMBA), azelaicbishydroxamic acid (ABHA), azelaic-1-hydroxamate-9-anilide (AAHA),6-(3-Chlorophenylureido) carpoic hydroxamic acid, or A-161906 asnon-limiting examples.

Hydroxyamic acid compounds having HDac inhibitory activity are describedin U.S. Pat. Nos. 6,800,638, 6,784,173, 6,531,472, 6,495,719, 6,512,123,and 6,511,990, U.S. Patent Publication Nos. 20060004041, 20050227976,20050187261, 20050107348, 20050131018, 20050124679, 20050085507,20040266818, 20040122079, 20040024067, and 20030018062, Intl.Publication Nos. EP1174438, WO/2004092115, WO/2005019174, WO0052033,WO018045, WO018171, WO0138322, WO0170675, WO9735990, WO9911659,WO0226703, WO0230879 and WO0226696, and in Butler et al, Clin CancerRes., 7: 962-970 (2001), Richon et al, Proc. Natl. Acad. Sci. USA: 95;3003-3007 (1998), Kim et al. Oncogene: 18(15); U.S. Pat. No. 24,612,470(1999), Klan et al, Biol Chem., 384(5):777-85 (2003), Yoshida et al, JBiol Chem., 265(28): 17174-9 (1990), Suzuli et al, Bioorg Med ChemLett., 15(2):331-5 (2005), Kelly et al, J Clin Oncol., 23(17):3923-31(2005), Kelly et al, Clin Cancer Res., 9(10 Pt 1):3578-88 (2003), Sonodaet al. Oncogene, 13(1):143-9 (1996), Richon et al, Proc Natl Acad SciUSA., 93(12):5705-8 (1996), Jung et al, J. Med. Chem., 42; 4669-4679.(1999), Jung et al, Bioorg. Med. Chem. Lett, 7(13); 1655-1658 (1997),Lavoie et al, Bioorg. Med. Chem. Letters 11, 2847-2850 (2001),Remiszewski et al, J. Med. Chem. 45, 4, 753-757 (2002), Sternson et al.Org. Lett. 3, 26, 4239-4242 (2001), Bouchain et al, J Med Chem.,46(5):820-30 (2003), and Woo et al, J Med Chem., 45(13):2877-85 (2002).

An HDAC inhibitor can be a cyclic tetrapeptide, such as Depsipeptide(FK228), FR225497, trapoxin A, apicidin, chlamydocin, or HC-toxin asnon-limiting examples. Cyclic tetrapeptides having HDAC inhibitoryactivity are described in U.S. Pat. Nos. 5,922,837, 6,403,555,6,656,905, 6,399,568, 6,825,317, 6,831,061, U.S. Patent Publication Nos.20050209134, 20040014647, 20030078369, and 20020120099, and in Kijima etal, J Biol Chem, 268(30):22429-35 (1993), Jose et al, Bioorg Med Chem Ze#, 14(21):5343-6 (2004), Xiao et al. Rapid Commun Mass Spectrom.,17(8):757-66 (2003), Furumai et al. Cancer Res., 62(17):4916-21 (2002),Nakajima et al, Exp. Cell Res., 241; 126-133 (1998), Sandor et al, ClinCancer Res., 8(3):718-28 (2002), Jung et al, J. Med. Chem., 42;4669-4679. (1999), and Jung et al, Bioorg. Med. Chem. Lett, 7(13);1655-1658 (1997).

An HDAC inhibitor can be a benzamide, such as MS-275. Benzamides havingHDAC inhibitory activity are described in U.S. Pat. Nos. 6,174,905 and6,638,530, U.S. Patent Publication Nos. 2004005513, 20050171103,20050131018, and 20040224991, Intl. Publication Nos. WO/2004082638,WO/2005066151, WO/2005065681, EP 0847992 and JP 258863/96, and in Saitoet al, Proc. Natl. Acad. Sci. USA, vol. 96, pp. 45924597 (1999); Suzukiet al, J. Med. Chem., vol. 42, pp. 3001-3003 (1999), Ryan et al, J ClinOncol, 23(17):391222 (2005), Pauer et al. Cancer Invest. 22(6):886-96(2004), and Undevia et al, Ann Oncol, 15(11): 1705-11 (2004).

An HDAC inhibitor can be a depudecin, a sulfonamide anilide (e.g.,diallyl sulfide), BL1521, curcumin (diferuloylmethane), CI-994(N-acetyldinaline), spiruchostatin A, Scriptaid, carbamazepine (CBZ), ora related compound. These and related compounds having HDac inhibitoryactivity are described in U.S. Pat. No. 6,544,957, and in Lea et al.Int. J. Oncol, 15, 347-352 (1999), Ouwehand et al, FEBS Lett.,579(6):1523-8 (2005), Kraker et al, Mol Cancer Ther. 2(4):401-8 (2003),de Ruijter et al, Biochem Pharmacol, 68(7): 1279-88 (2004), Liu et al.Acta Pharmacol Sin., 26(5):603-9 (2005), Fournel et al. Cancer Res., 62:4325-4330 (2002), Yurek-George et al, J Am Chem Soc, 126(4):1030-1(2004), Su et al. Cancer Res., 60(12):3137-42 (2000), Beutler et al.Life Sci., 76(26):3107-15 (2005), and Kwon et al, Proc. Natl. Acad. Sci.USA 95, 3356-3361 (1998).

An HDAC inhibitor can be a compound comprising a cyclic tetrapeptidegroup and a hydroxamic acid group. Examples of such compounds aredescribed in U.S. Pat. Nos. 6,833,384 and 6,552,065, and in Nishino etal, Bioorg Med Chem., 12(22):5777-84 (2004), Nishino et al. Org Lett,5(26):5079-82 (2003), Komatsu et al. Cancer Res., 61(11):4459-66 (2001),Furumai et al, Proc Natl Acad Sci USA., 98(1):87-92 (2001), Yoshida etal. Cancer Chemotherapy and Pharmacology, 48 Suppl. 1; S20-S26 (2001),and Remiszeski et al, J Med Chem., 46(21):4609-24 (2003).

An HDAC inhibitor can be a compound comprising a benzamide group and ahydroxamic acid group. Examples of such compounds are described in Ryuet al. Cancer Lett. Jul. 9, 2005 (epub), Plumb et al, Mol Cancer Ther,2(8):721-8 (2003), Ragno et al, J Med Chem., 47(6):1351-9 (2004), Mai etal, J Med Chem., 47(5):1098109 (2004), Mai et al, J Med Chem.,46(4):512-24 (2003), Mai et al, J Med Chem., 45(9):1778-84 (2002), Massaet al, J Med Chem., 44(13):2069-72 (2001), Mai et al, J Med Chem.,48(9):3344-53 (2005), and Mai et al, J Med Chem., 46(23):4826-9 (2003).

An HDAC inhibitor can be a compound described in U.S. Pat. Nos.6,897,220, 6,888,027, 5,369,108, 6,541,661, 6,720,445, 6,562,995,6,777,217, or 6,387,673, 6,693,132, or U.S. Patent Publication Nos.20060020131, 20060058553, 20060058298, 20060058282, 20060052599,2006004712, 20060030554, 20060030543, 20050288282, 20050245518,20050148613, 20050107348, 20050026907, 20040214880, 20040214862,20040162317, 20040157924, 20040157841, 20040138270, 20040072849,20040029922, 20040029903, 20040023944, 20030125306, 20030083521,20020143052, 20020143037, 20050197336, 20050222414, 20050176686,20050277583, 20050250784, 20050234033, 20050222410, 20050176764,20050107290, 20040043470, 20050171347, 20050165016, 20050159470,20050143385, 20050137234, 20050137232, 20050119250, 20050113373,20050107445, 20050107384, 20050096468, 20050085515, 20050032831,20050014839, 20040266769, 20040254220, 20040229889, 20040198830,20040142953, 20040106599, 20040092598, 20040077726, 20040077698,20040053960, 20040002506, 20030187027, 20020177594, 20020161045,20020119996, 20020115826, 20020103192, or 20020065282.

An HDAC inhibitor can be inhibitor is selected from the group consistingof FK228, AN-9, MS-275, CI-994, LAQ-824, SAHA, G2M-777, PXD-101,LBH-589, MGCD-0103, MK0683, pyroxamide, sodium phenylbutyrate,CRA-024781, and derivatives, salts, metabolites, prodrugs, andstereoisomers thereof.

Additional non-limiting examples include a reported HDAC inhibitorselected from ONO-2506 or arundic acid (CAS RN 185517-21-9); MGCD0103(see Gelmon et al. “Phase I trials of the oral histone deacetylase(HDac) inhibitor MGCD0103 given either daily or 3× weekly for 14 daysevery 3 weeks in patients (pts) with advanced solid tumors.” Journal ofClinical Oncology, 2005 ASCO Annual Meeting Proceedings. 23(16S, June 1Supplement), 2005: 3147 and Kalita et al. “Pharmacodynamic effect ofMGCD0103, an oral isotype-selective histone deacetylase (HDac)inhibitor, on HDac enzyme inhibition and histone acetylation inductionin Phase I clinical trials in patients (pts) with advanced solid tumorsor non-Hodgkin's lymphoma (NHL)” Journal of Clinical Oncology, 2005 ASCOAnnual Meeting Proceedings. 23(16S, Part I of II, June 1 Supplement),2005: 9631), a reported thiophenyl derivative of benzamide HDacinhibitor as presented at the 97th American Association for CancerResearch (AACR) Annual Meeting in Washington, D.C. in a poster titled“Enhanced Isotype-Selectivity and Antiproliferative Activity ofThiophenyl Derivatives of BenzamideHDac Inhibitors In Human CancerCells,” (abstract #4725), and a reported HDac inhibitor as described inU.S. Pat. No. 6,541,661; SAHA or Vorinostat (CAS RN 149647-78-9); PXD101or PXD 101 or PX 105684 (CAS RN 414864-00-9), CI-994 or Tacedinaline(CAS RN 112522-64-2), MS-275 (CAS RN 209783-80-2), or an inhibitorreported in WO2005/108367.

An HDAC inhibitor can be a novel HDac inhibitor identified usingstructure-activity relationships and teachings known in the art anddescribed, e.g., in Miller et al., J. Med. Chem., 46(24); 5097-5115(2003) and Klan et al., Biol Chem., 384(5):777-85 (2003)), all of whichare incorporated herein by reference in their entirety. Methods toassess histone deacetylase activity are known in the art, and aredescribed, e.g., in Richon et al., Methods Enzymol, 376:199-205 (2004),Wegener et al., Mol Genet Metab., 80(1-2): 138-47 (2003), U.S. Pat. No.6,110,697, and U.S. Patent Publication Nos. 20050118596, 20050227300,20030161830, 20030224473, 20030082668, 20030013176, and 20040091951),all of which are incorporated herein by reference in their entirety.

Antisense oligonucleotides and ribozymes that inhibit transcriptionand/or translation of one or more HDacs are described in U.S. Pat. No.6,953,783, and U.S. Patent Publication Nos. 20050171042, 20040266718,20040204373, 20040077578, 20040077084, 20040077083, 20040072770,20030236204, 20030216345, 20030152557, 20030148970, 20030078216,20020137162, 20020164752, 20020115177, and 20020061860.

Some exemplary inhibitors of HDAC include small molecular weightcarboxylates (e.g., less than about 250 amu), hydroxamic acids,benzamides, epoxyketones, cyclic peptides, and hybrid molecules. (See,for example, Drummond D. C., et al. Annu. Rev. Pharmacol. Toxicol.(2005) 45: 495-528, (including specific examples therein) which ishereby incorporated by reference in its entirety). Non-limiting examplesHDAC inhibitors include, but are not limited to, SuberoylanilideHydroxamic Acid (SAHA (e.g., MK0683, vorinostat) and other hydroxamicacids), BML-210, Depudecin (e.g., (−)-Depudecin), HC Toxin, Nullscript(4-(1,3-Dioxo-1H,3H-benzo[de]isoquinolin-2-yl)-N-hydroxybutanamide),Phenylbutyrate (e.g., sodium phenylbutyrate) and Valproic Acid ((VPA)and other short chain fatty acids), Scriptaid, Suramin Sodium,Trichostatin A (TSA), APHA Compound 8, Apicidin, Sodium Butyrate,pivaloyloxymethyl butyrate (Pivanex, AN-9), Trapoxin B, Chlamydocin,Depsipeptide (also known as FR901228 or FK228), benzamides (e.g., CI-994(i.e., N-acetyl dinaline) and MS-27-275), MGCD0103, NVP-LAQ-824, CBHA(m-carboxycinnaminic acid bishydroxamic acid), JNJ16241199, Tubacin,A-161906, proxamide, oxamflatin, 3-Cl-UCHA (i.e.,6-(3-chlorophenylureido)caproic hydroxamic acid), AOE(2-amino-8-oxo-9,10-epoxydecanoic acid), CHAP31 and CHAP 50. Otherinhibitors include, for example, dominant negative forms of the HDACs(e.g., catalytically inactive forms) siRNA inhibitors of the HDACs, andantibodies that specifically bind to the HDACs. HDAC inhibitors arecommercially available, e.g., from BIOMOL International, Fukasawa, MerckBiosciences, Novartis, Gloucester Pharmaceuticals, Aton Pharma, TitanPharmaceuticals, Schering AG, Pharmion, MethylGene, and Sigma Aldrich.Further HDAC inhibitors amenable to the invention include, but are notlimited to, those that are described in U.S. Pat. Nos. 7,183,298;6,512,123; 6,541,661; 6,531472; 6,960,685; 6,897,220; 6,905,669;6,888,207; 6,800,638 and 7,169,801, and U.S. patent application Ser.Nos. 10/811,332; 12/286,769; 11/365,268; 11/581,570; 10/509,732;10/546,153; 10/381,791 and 11/516,620, the contents of which each areincorporated herein by reference in their entirety.

In some embodiments, the HDAC modulator inhibits binding of a ligand byat least about or about any one of 10%, 20%, 30%, 40%, 50%, 60%, 70%,80%, 90%, 95% or 100% relative to a control.

In some embodiments, the HDAC modulator reduces an activity of a HDAC byat least 5%, at least 10%, at least 20%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100% (e.g. complete loss of activity)relative to an uninhibited control.

In some embodiments, the HDAC modulator enhances an activity of a HDACby at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold,1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold or more relative toan unactivated control.

In some embodiments, the HDAC modulator is capable of binding to theactive site of a HDAC (e.g., a binding site for a ligand).

In some embodiments, the HDAC modulator is capable of binding to anallosteric site of a HDAC.

In some embodiments of this and other aspects of the invention, the HDACantagonist has an IC50 of less than or equal to 500 nM, less than orequal to 250 nM, less than or equal to 100 nM, less than or equal to 50nM, less than or equal to 10 nM, less than or equal to 1 nM, less thanor equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equalto 0.001 nM.

In some embodiments of this and other aspects of the invention, the HDACagonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

Neuropeptides

Neuropeptides are small protein-like molecules used by neurons tocommunicate with each other, distinct from the larger neurotransmitters.They are neuronal signaling molecules, influence the activity of thebrain in specific ways and are thus involved in particular brainfunctions, like analgesia, reward, food intake, learning and memory.Neuropeptides are expressed and released by neurons, and mediate ormodulate neuronal communication by acting on cell surface receptors. Thehuman genome contains about 90 genes that encode precursors ofneuropeptides. At present about 100 different peptides are known to bereleased by different populations of neurons in the mammalian brain.

Exemplary neuropeptides include, but are not limited to, hypothalamichormones such as oxytocin and vasopressin; hypothalamic releasing andinhibiting hormones such as corticotropin releasing hormone (CRH),growth hormone releasing hormone (GHRH), luteinizing hormone releasinghormone (LHRH), somatostatin growth hormone release inhibiting hormoneand thyrotropin releasing hormone; tachykinins such as neurokinin a(substance K), neurokinin b, neuropeptide K and substance P; opioidpeptides such as b-endorphin, dynorphin and met- and leu-enkephalin; NPYand related peptides such as neuropeptide tyrosine (NPY), pancreaticpolypeptide and peptide tyrosine-tyrosine (PYY); VIP-glucagon familymembers such as glucogen-like peptide-1 (GLP-1), peptide histidineisoleucine (PHI), pituitary adenylate cyclase activating peptide (PACAP)and vasoactive intestinal polypeptide (VIP); as well as many otherpeptides such as brain natriuretic peptide), calcitonin gene-relatedpeptide (CGRP) (a- and b-form), cholecystokinin (CCK) and other forms,galanin, islet amyloid polypeptide (LAPP) or amylin, melaninconcentrating hormone (MCH), melanocortins (ACTH, a-MSH and others),neuropeptide FF (F8Fa), neurotensin, parathyroid hormone relatedprotein, Agouti gene-related protein (AGRP), cocaine and amphetamineregulated transcript (CART)/peptide, endomorphin-1 and -2,5-HT-moduline, hypocretins/orexins, nociceptin/orphanin FQ, nocistatin,prolactin releasing peptide, secretoneurin and urocortin; Neurotensin;Neuropeptide Y; Neurotensin; Substance P; TRH; Enkephalin; and the like

In some embodiments, the neuropeptide can PD160170

Ionophores

As used herein, the term “ionophore” includes molecules capable offorming a complex with a particular ion, in some instances to thesubstantial exclusion of others. Generally, an ionophore facilitatestransmission of an ion across a lipid barrier by combining with the ionor by increasing the permeability of the barrier to it.

Without limitations, an ionophore can be selected from the groupconsisting of small or large organic or inorganic molecules;monosaccharides; disaccharides; trisaccharides; oligosaccharides;polysaccharides; biological macromolecules, e.g., proteins, peptides,peptide analogs and derivatives thereof, peptidomimetics, nucleic acids,nucleic acid analogs and derivatives, enzymes, antibodies, portion orfragments of antibodies; an extract made from biological materials suchas bacteria, plants, fungi, or animal cells or tissues; naturallyoccurring or synthetic compositions; and any combinations thereof.

Exemplary potassium ion ionophores include, but are not limited to,valinomycin, crown ethers, e.g., dimethyldibenzo-30-crown-10,dicyclohexyl-18-crown, dimethyldicyclohexyl-18-crown-6, tetraphenylborate, tetrakis(chlorophenyl)borate. Sodium ion ionophores include, forexample, methyl monensin,N,N′,N″-triheptyl-N,N′,N″trimethyl-4,4′,4″-propylidintris-(3-oxabutyramide),N,N,N,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide,4-octadecanoyloxymethyl-N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide,bis [(12-crown-4)methyl] dodecylmethylmalonate. Exemplary calcium ionionophores include, but are not limited to, bis(didecylphosphate),bis(4-octylphenylphosphate), bis(4-(1,1,3,3-tetramethylbutyl)phenylphosphatetetracosamethylcyclododecasiloxane,N,N′-di(1,1,3,3ethoxycarbonyl)undecyl)-N,N′,4,5-tetramethyl-3,6dioxaoctane diamide, calcium ionophore A23187 (alsoknown as C-7522), calcium ionophore II 21193, and calcium ionophore IV21198. Exemplary barium ion ionophores include, but are not limited to,calcium di(2-ethylhexyl)phosphate+decan-1-ol, barium complex ofnonylphenoxypoly(ethyleneoxy)ethanol in ortho-nitrodiphenyl ether.Exemplary chloride ion ionophores include, but are not limited to,{u-[4,5-dimethyl-3,6-bis(octyloxy)-1,2-phenylene]}bis(trifluoroacetato-0)dimercuri (ETH 9009),{(x-[4,5-dimethyl-3,6-bis(dodecyloxy)-1,2-phenylene]}bis (mercurychloride) (ETH 9033), 5,10,15,20-tetraphenyl21H,23H-porphin manganese(III) chloride (MnTPPCl), tributyltin chloride (TBTC1) and trioctyltinchloride (TOTC1). Bicarbonate ion ionophores include, for example,quaternary ammonium ion exchangerp-octodecyloxy-meta-chlorophenyl-hydrazone-mesoxalonitrile. Ammonium ionionophores include, for example, nonactin and monactin. Nitrate ionionophores include, for example, tridodecylhexadecylammoniumnitrate+n-octylortho-nitrophenyl, 1:10 phenanthroline nickel (II)nitrate+ para-nitrocymene. Lithium ion ionophores include, for example,N,N′-diheptyl-N,N′,5,5-tetramethyl-3,7-dioxononanediamide),12-crown-4,6,6-dibenzyl-14-crown-4. Another non-limiting exemplary listof ionophores includes: for potassium, valinomycin,dicyclohexano-18-crown-6, dibenzo-18-crown-6, tetraphenyl borate,tetrakis (chlorophenyl) borate; for calcium, bis(didecylphosphate),bis(4-octylphenylphosphate), bis(4-(1,1,3,3-tetramethylbutyl)phenylphosphate tetracosamefhylcyclododecasiloxane, N,N′-di(ll-ethoxycarbonyl) undecyl)-N, N′,4,5-tetramethyl-3,6-dioxaoctanediamide; for hydrogen, tridodecylamine, N-methyl N-octadecyl (1-methyl,2-hydroxy, 2-phenyl) ethylamine, N-octadecyl 3-hydroxy n-propylamine, N,N′ bis (octadecyl ethylene amine),p-octadecyloxy-m-chlorophenylhydrazonemeso oxalonitrile; for sodium,monensin, N,N′,N″-triheptyl-N, N, N″-trimethyl-4, 4′,4″-propylidintris-(3-oxabutyramide),N,N,N′,N′-tetracyclohexyl-1,2-phenylenedioxydiacetamide,4-octadecanoyloxymethyl-N,N,N′,N′,-tetracyclohexyl-1,2-phenylenedioxydiacetamide,bis[(12-crown-4)methyl] dodecylmethylmalonate; for lithium, N,N′-diheptyl-N, N, 5,5-tetramethyl-3,7-dioxononanediamide), 12-crown-4,6,6dibenzyl-14 crown-4; for chloride, quaternary ammonium chloride,tributyl tin chloride. Other suitable ionophores include ionomycin,monensin, lasalocid, laidlomycin, and the like

Ion Channel Modulators

As used herein, the term “ion-channel modulator” refers to a compoundthat modulates at least one activity of an ion-channel. The term“ion-channel modulator” as used herein is intended to include agentsthat interact with the channel pore itself, or that may act as anallosteric modulator of the channel by interacting with a site on thechannel complex. The term “ion-channel modulator” as used herein is alsointended to include agents that modulate activity of an ion-channelindirectly. By “indirectly,” as used in reference to modulatorinteractions with ion-channel, means the ion-channel modulator does notdirectly interact with the ion-channel itself, i.e., ion-channelmodulator interacts with the ion-channel via an intermediary.Accordingly, the term “indirectly” also encompasses the situationswherein the ion-channel modulator requires another molecule in order tobind or interact with the ion-channel.

Without limitations, an ion-channel modulator can be selected from thegroup consisting of small or large organic or inorganic molecules;monosaccharides; disaccharides; trisaccharides; oligosaccharides;polysaccharides; biological macromolecules, e.g., proteins, peptides,peptide analogs and derivatives thereof, peptidomimetics, nucleic acids,nucleic acid analogs and derivatives, enzymes, antibodies, portion orfragments of antibodies; an extract made from biological materials suchas bacteria, plants, fungi, or animal cells or tissues; naturallyoccurring or synthetic compositions; and any combinations thereof.

In some embodiments of the aspects described herein, the ion-channelmodulator modulates the passage of ions through the ion-channel.

In some embodiments of the aspects described herein, the modulator is aninhibitor or antagonist of the ion-channel. As used herein, the term“inhibitor” refers to compounds which inhibit or decrease the flow ofions through an ion-channel.

In some embodiments of the aspects described herein, the modulator is anagonist of the ion-channel. As used herein, the term “agonist” refers tocompounds which increase the flow of ions through an ion-channel.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the ion-channel by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, at least 98% or more relative to a controlwith no modulation.

In some embodiments of the aspects described herein, at least oneactivity of the ion-channel is inhibited or lowered by at least 5%, atleast 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, at least 95%, at least 98%, or 100% (e.g. complete loss ofactivity) relative to control with no modulator.

In some embodiments of the aspects described herein, the ion-channelmodulator has an IC₅₀ of less than or equal to 500 nM, 250 nM, 100 nM,50 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

In some embodiments of the aspects described herein, the ion-channelmodulator inhibits the flow of ions through the ion-channel by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 95%, at least 98%, or 100% (e.g. completestop of ion flow through the channel) relative to a control with nomodulator.

In some embodiments of the aspects described herein, the ion-channelmodulator increases the flow of ions through the ion-channel by at least5%, at least 10%, at least 15%, at least 20%, at least 25%, at least30%, at least 40%, at least 50%, at least 60%, at least 70%, at least80%, at least 90%, at least 100%, at least 1.5 fold, at least by 2-fold,at least 3-fold, at least 4-fold, or at least 5-fold or more relative toa control with no modulator.

In some embodiments of the aspects described herein, the ion-channelmodulator increases concentration of ions, e.g. sodium, in a cell by atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 100%, at least 1.5 fold, at least by2-fold, at least 3-fold, at least 4-fold, or at least 5-fold or morerelative to a control with no modulator.

Without wishing to be bound by a theory, an ion-channel modulator canmodulate the activity of an ion-channel through a number of differentmechanisms. For example, a modulator can bind with the ion-channel andphysically block the ions from going through the channel. An ion-channelmodulator can bring about conformational changes in the ion-channel uponbinding, which may increase or decrease the interaction between the ionsand the channel or may increase or decrease channel opening.

A modulator can modulate the energy utilizing activity, e.g. ATPaseactivity, of the ion-channel. In some embodiments of the aspectsdescribed herein, the ion-channel modulator inhibits the ATPAse activityof the ion-channel.

In some embodiments of the aspects described herein, an ion-channelmodulator inhibits ATPase activity of the Na⁺/K⁺-ATPase by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 75%, atleast 80%, at least 85%, at least 90%, at least 95%, at least 98%, or100% (complete inhibition) relative to a control without the modulator.Without wishing to be bound by theory, ATPase activity can be measuredby measuring the dephosphorylation of adenosine-triphosphate byutilizing methods well known to the skilled artisan for measuring suchdephosphorylation reactions.

In some embodiments of the aspects described herein, an ion-channelmodulator inhibits RIG-I activation by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 75%, at least 80%, atleast 85%, at least 90%, at least 95%, at least 98%, or 100% (completeinhibition) relative to a control without the modulator.

In some embodiments of the aspects described herein, an ion-channelmodulator inhibits ATPase activity of RIG-I by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 75%, at least80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%(complete inhibition) relative to a control without the modulator.

Without limitation, the ion-channel modulator can be a small organicmolecule, small inorganic molecule, a polysaccharide, a peptide, aprotein, a nucleic acid, an extract made from biological materials suchas bacteria, plants, fungi, animal cells, animal tissue, and anycombinations thereof.

In some embodiments of the aspects described herein, the ion-channelmodulator is a cardiac glycoside. As used herein, the term “cardiacglycoside” refers to the category of compounds that have a positiveinotropic effect on the heart. Cardiac glycosides are also referred toas cardiac steroids in the art. They are used in treatment of heartdiseases, including cardiac arrhythmia and have a rate dependent effectupon AV nodal conduction. As a general class of compounds, cardiacglycosides comprise a steroid core with either a pyrone or butenolidesubstituent at C17 (the “pyrone form” and “butenolide form”).Additionally, cardiac glycosides may optionally be glycosylated at C3.The form of cardiac glycosides without glycosylation is also known as“aglycone.” Most cardiac glycosides include one to four sugars attachedto the 3R—OH group. The sugars most commonly used include L-rhamnose,D-glucose, D-digitoxose, D-digitalose, D-digginose, D-sarmentose,L-vallarose, and D-fructose. In general, the sugars affect thepharmacokinetics of a cardiac glycoside with little other effect onbiological activity. For this reason, aglycone forms of cardiacglycosides are available and are intended to be encompassed by the term“cardiac glycoside” as used herein. The pharmacokinetics of a cardiacglycoside may be adjusted by adjusting the hydrophobicity of themolecule, with increasing hydrophobicity tending to result in greaterabsorption and an increased half-life. Sugar moieties may be modifiedwith one or more groups, such as an acetyl group.

A large number of cardiac glycosides are known in the art. Exemplarycardiac glycoside include, but are not limited to, bufalin, ouabain,digitoxigenin, digoxin, lanatoside C, Strophantin K, uzarigenin,desacetyllanatoside A, digitoxin, actyl digitoxin, desacetyllanatosideC, strophanthoside, scillarenin, scillaren A, proscillaridin,proscillaridin A, BNC-1, BNC-4, digitoxose, gitoxin, strophanthidiol,oleandrin, acovenoside A, strophanthidine digilanobioside,strophanthidin-d-cymaroside, digitoxigenin-L-rhamnoside, digitoxigenintheretoside, strophanthidin, strophanthidine, strophanthidinedigilanobioside, strophanthidin-Dcymaroside, digoxigenin, digoxigenin3,12-diacetate, gitoxigenin, gitoxigenin 3-acetate, gitoxigenin3,16-diacetate, 16-acetyl gitoxigenin, acetyl strophanthidin,ouabagenin, 3-epigoxigenin, neriifolin, acetylneriifolin cerberin,theventin, somalin, odoroside, honghelin, desacetyl digilanide,calotropin, calotoxin, lanatoside A, uzarin,strophanthidine-3β-digitoxoside, strophanthidin a-L-rhamnopyranoside,and analogs, derivatives, pharmaceutically acceptable salts, and/orprodrugs thereof.

More than a hundred cardiac glycosides have been identified as secondarymetabolites in plants, with most belonging to the angiosperms. See forexample, Melero, C. P., Medardea, M. & Feliciano, A. S. A short reviewon cardiotonic steroids and their aminoguanidine analogues. Molecules 5,51-81 (2000), content of which is herein incorporated by reference.Generally, cardiac glycosides are found in a diverse group of plantsincluding Digitalis purpurea and Digitalis lanata (foxgloves), Neriumoleander (common oleander), Thevetia peruviana (yellow oleander),Convallaria majalis (lily of the valley), Urginea maritima and Urgineaindica (squill), and Strophanthus gratus (ouabain). Recently, however,cardiac glycosides of the bufadienolide class were identified in theskin and the carotid gland of animals, and mainly in the venom ofseveral toad species. See Steyn, P. S. & van Heerden, F. R.Bufadienolides of plant and animal origin. Nat. Prod. Rep. 15, 397-413(1998), content of which is herein incorporated by reference.

In some embodiments of the aspects described herein, the ion-channelmodulator is a sodium pump blocker. As used herein, the terms “sodiumpump blocker,” “sodium pump inhibitor,” and “sodium pump antagonist”refer to compounds that inhibit or block the flow of sodium and/orpotassium ions across a cell membrane.

In some embodiments of the aspects described herein, the ion-channelmodulator is a calcium channel blocker. As used herein, the terms“calcium channel blocker,” “calcium channel inhibitor,” and “calciumchannel antagonist” refer to compounds that inhibit or block the flow ofcalcium ions across a cell membrane. Calcium channel blockers are alsoknown as calcium ion influx inhibitors, slow channel blockers, calciumion antagonists, calcium channel antagonist drugs and as class IVantiarrhythmics. Exemplary calcium channel blocker include, but are notlimited to, amiloride, amlodipine, bepridil, diltiazem, felodipine,isradipine, mibefradil, nicardipine, nifedipine (dihydropyridines),nickel, nimodinpine, nisoldipine, nitric oxide (NO), norverapamil,verapamil, and analogs, derivatives, pharmaceutically acceptable salts,and/or prodrugs thereof.

In some embodiments of the aspects described herein, the calcium channelblocker is a beta-blocker. Exemplary beta-blockers include, but are notlimited to, Alprenolol, Bucindolol, Carteolol, Carvedilol (hasadditional α-blocking activity), Labetalol, Nadolol, Penbutolol,Pindolol, Propranolol, Timolol, Acebutolol, Atenolol, Betaxolol,Bisoprolol, Celiprolol, Esmolol, Metoprolol, Nebivolol, Butaxamine, andICI-118,551 (3-(isopropylamino)-1-[(7-methyl-4-indanyl)oxy]butan-2-ol),and analogs, derivatives, pharmaceutically acceptable salts, and/orprodrugs thereof.

Exemplary K⁺ ion-channel modulators include, but are not limited to,2,3-Butanedione monoxime; 3-Benzidino-6-(4-chlorophenyl)pyridazine;4-Aminopyridine; 5-(4-Phenoxybutoxy)psoralen; 5-Hydroxydecanoic acidsodium salt; L-α-Phosphatidyl-D-myo-inositol; 4,5-diphosphate,dioctanoyl; Aa1; Adenosine 5′-(β,γ-imido)triphosphate tetralithium salthydrate; Agitoxin-1; Agitoxin-2; Agitoxin-3; Alinidine; Apamin;Aprindine hydrochloride; BDS-I; BDS-II; BL-1249; BeKm-1; CP-339818;Charybdotoxin; Charybdotoxin; Chlorzoxazone; Chromanol 293B; Cibenzolinesuccinate; Clofilium tosylate; Clotrimazole; Cromakalim; CyPPA; DK-AH269; Dendrotoxin-I; Dendrotoxin-K; Dequalinium chloride hydrate; DPO-1needles; Diazoxide; Dofetilide; E-4031; Ergtoxin; Glimepiride;Glipizide; Glybenclamide; Heteropodatoxin-2; Hongotoxin-1; ICA-105574;IMID-4F hydrochloride; Iberiotoxin; Ibutilide hemifumarate salt;Isopimaric Acid; Kaliotoxin-1; Levcromakalim; Lq2; Margatoxin; Mast CellDegranulating Peptide; Maurotoxin; Mephetyl tetrazole; Mepivacainehydrochloride; Minoxidil; Minoxidil sulfate salt; N-Acetylprocainamidehydrochloride; N-Salicyloyltryptamine; NS 1619; NS1643; NS309; NS8593hydrochloride; Nicorandil; Noxiustoxin; Omeprazole; PD-118057;PNU-37883A; Pandinotoxin-Kα; Paxilline; Penitrem A; Phrixotoxin-2;Pinacidil monohydrate; Psora-4; Quinine; Quinine hemisulfate saltmonohydrate; Quinine hydrobromide; Quinine hydrochloride dehydrate;Repaglinide; Rutaecarpine; S(+)-Niguldipine hydrochloride; SG-209;Scyllatoxin; Sematilide monohydrochloride monohydrate; Slotoxin;Stromatoxin-1; TRAM-34; Tamapin; Tertiapin; Tertiapin-Q trifluoroacetatesalt; Tetracaine; Tetracaine hydrochloride; Tetraethylammonium chloride;Tityustoxin-Kα; Tolazamide; UCL 1684; UCL-1848 trifluoroacetate salt;UK-78282 monohydrochloride; VU 590 dihydrochloride hydrate; XE-991;ZD7288 hydrate; Zatebradine hydrochloride; α-Dendrotoxin; β-Dendrotoxin;δ-Dendrotoxin; γ-Dendrotoxin; β-Bungarotoxin; and analogs, derivatives,pharmaceutically acceptable salts, and/or prodrugs thereof.

In some embodiments of the aspects described herein, the ion-channelmodulator is a potassium channel agonist. As used herein, a “potassiumchannel agonist” is a K⁺ ion-channel modulator which facilitates iontransmission through K⁺ ion-channels. Exemplary potassium channelagonists include, but are not limited to diazoxide, minoxidil,nicorandil, pinacidil, retigabine, flupirtine, lemakalim, L-735534, andanalogs, derivatives, pharmaceutically acceptable salts, and/or prodrugsthereof.

In some embodiments of the aspects described herein, the ion-channelmodulator is selected from the group consisting of bufalin; digoxin;ouabain; nimodipine; diazoxide; digitoxigenin; ranolazine; lanatoside C;Strophantin K; uzarigenin; desacetyllanatoside A; actyl digitoxin;desacetyllanatoside C; strophanthoside; scillaren A; proscillaridin A;digitoxose; gitoxin; strophanthidiol; oleandrin; acovenoside A;strophanthidine digilanobioside; strophanthidin-d-cymaroside;digitoxigenin-L-rhamnoside; digitoxigenin theretoside; strophanthidin;digoxigenin-3,12-diacetate; gitoxigenin; gitoxigenin 3-acetate;gitoxigenin-3,16-diacetate; 16-acetyl gitoxigenin; acetylstrophanthidin; ouabagenin; 3-epigoxigenin; neriifolin; acetyhieriifolincerberin; theventin; somalin; odoroside; honghelin; desacetyldigilanide; calotropin; calotoxin; convallatoxin; oleandrigenin;periplocyrnarin; strophanthidin oxime; strophanthidin semicarbazone;strophanthidinic acid lactone acetate; ernicyrnarin; sannentoside D;sarverogenin; sarmentoside A; sarmentogenin; proscillariditi;marinobufagenin; Amiodarone; Dofetilide; Sotalol; Ibutilide; Azimilide;Bretylium; Clofilium;N-[4-[[1-[2-(6-Methyl-2-pyridinyl)ethyl]-4-piperidinyl]carbonyl]phenyl]methanesulfonamide(E-4031); Nifekalant; Tedisamil; Sematilide; Ampyra; apamin;charybdotoxin; 1-Ethyl-2-benzimidazolinone (1-EBIO);3-Oxime-6,7-dichloro-1H-indole-2,3-dione (NS309);Cyclohexyl-[2-(3,5-dimethyl-pyrazol-1-yl)-6-methyl-pyrimidin-4-yl]-amine(CyPPA); GPCR antagonists; ifenprodil; glibenclamide; tolbutamide;diazoxide; pinacidil; halothane; tetraethylammonium; 4-aminopyridine;dendrotoxins; retigabine; 4-aminopyridine; 3,4-diaminopyridine;diazoxide; Minoxidil; Nicorandi; Retigabine; Flupirtine; Quinidine;Procainamide; Disopyramide; Lidocaine; Phenytoin; Mexiletine;Flecainide; Propafenone; Moricizine; atenolol; ropranolol; Esmolol;Timolol; Metoprolol; Atenolol; Bisoprolol; Amiodarone; Sotalol;Ibutilide; Dofetilide; Adenosine; Nifedipine; δ-conotoxin; κ-conotoxin;ω-conotoxin; ω-conotoxin; ω-conotoxin GVIA; ω-conotoxin ω-conotoxinCNVIIA; ω-conotoxin CVIID; ω-conotoxin AM336; cilnidipine; L-cysteinederivative 2A; ω-agatoxin IVA; N,N-dialkyl-dipeptidyl-amines; SNX-111(Ziconotide); caffeine; lamotrigine; 202W92 (a structural analog oflamotrigine); phenytoin; carbamazepine;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 1-phenylethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methyl-2-propynyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, cyclopropylmethyl ester;1,4-dihydro-2,6-dimethyl-5-nitro-4-[thieno(3,2-c)pyridin-3-yl]-3-pyridinecarboxylicacid, butyl ester;(S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, methyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 2-propynyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methyl-2propynyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 2-butynyl este;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methyl-2butynyl este;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 2,2-dimethylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 3-butynyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1,1-dimethyl-2propynyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno3,2-c]pyridin-3-yl-3-pyridinecarboxylicacid, 1,2,2-trimethylpropyl ester;R(+)-1,4-Dihydro-2,6-dimethyl-5-nitro-4[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylic(2Amethyl-1-phenylpropyl) ester;S-(−)-1,4-Dihydro-2,6-dimethyl-5-nitro-4[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 2-methyl-1-phenylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methylphenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-phenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, (1-phenylpropyl)ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, (4-methoxyphenyl)methyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 1-methyl-2phenylethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 2-phenylpropyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, phenylmethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 2-phenoxyethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 3-phenyl-2propynyl este;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 2-methoxy2-phenylethyl ester;(S)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1phenylethyl este;(R)-1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1phenylethyl este;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, cyclopropylmethyl ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-thieno[3,2-c]pyridin-3-yl]-3-pyridinecarboxylicacid, 1-cyclopropylethyl este;1,4-Dihydro-2,6-dimethyl-5-nitro-4-[thieno[3,2c]-pyridin-3-yl]-3-pyridinecarboxylicacid, 2-cyanoethyl ester;1,4-Dihydro-4-(2-{5-[4-(2-methoxyphenyl)-1-1piperazinyl]pentyl}-3-furanyl)-2,6-dimethyl-5-nitro3-pyridinecarboxylicacid, methyl ester;4-(4-Benzofurazanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylicacid, {4-[4-(2-methoxyphenyl)-1-piperazinyl]butyl} ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-(3-pyridinyl)-3-pyridinecarboxylicacid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;4-(3-Furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3pyridinecarboxylic acid,{2-[4-(2-methoxyphenyl)-1piperazinyl]ethyl} ester;4-(3-Furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3pyridinecarboxylic acid,{2-[4-(2-pyrimidinyl)-1piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl)-5-nitro-3-pyridinecarboxylicacid, {4-[4-(2-methoxyphenyl) 1-piperazinyl]butyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2yl)-5-nitro-3-pyridinecarboxylicacid, {4-[4-(2pyrimidinyl)-1-piperazinyl]butyl} ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-(3-thienyl)-3-pyridinecarboxylicacid, {2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-(3-thienyl)-3-pyridinecarboxylicacid, {2-[4-(2-pyrimidinyl)-1-piperazinyl]ethyl} ester;4-(3-Furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylicacid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;(4-(2-Furanyl)-1,4-dihydro-2,6-dimethyl-5-nitro-3-pyridinecarboxylicacid, {4-[4-(2-pyrimidinyl)-1-piperazinyl]butyl} ester;1,4-Dihydro-2,6-dimethyl-5-nitro-4-(2-thienyl)-3-pyridinecarboxylicacid, {2-[4-(2-methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl)-5-nitro-3-pyridinecarboxylicacid, {2-[4-(2methoxyphenyl)-1-piperazinyl]ethyl} ester;1,4-Dihydro-2,6-dimethyl-4-(1-methyl-1H-pyrrol-2-yl)-5-nitro-3-pyridinecarboxylicacid, {2-[4-(2pyrimidinyl) 1-piperazinyl]ethyl} ester;5-(4-Chlorophenyl)-N-(3,5-dimethoxyphenyl)-2-furancarboxamide(A-803467); and analogs, derivatives, pharmaceutically acceptable salts,and/or prodrugs thereof.

In some embodiments of the aspects described herein, the ion-channelmodulator is bufalin or analogs, derivatives, pharmaceuticallyacceptable salts, and/or prodrugs thereof. Exemplary bufalin analogs andderivatives include, but are not limited to, 7β-Hydroxyl bufalin;3-epi-7β-Hydroxyl bufalin; 1β-Hydroxyl bufalin; 15α-Hydroxyl bufalin;15β-Hydroxyl bufalin; Telocinobufagin (5-hydroxyl bufalin);3-epi-Telocinobufagin; 3-epi-Bufalin-3-O-β-d-glucoside; 1 IP-Hydroxylbufalin; 12β-Hydroxyl bufalin; 1β,7β-Dihydroxyl bufalin; 16α-Hydroxylbufalin; 7β,16α-Dihydroxyl bufalin; 1β,12β-Dihydroxyl bufalin;resibufogenin; norbufalin;3-hydroxy-14(15)-en-19-norbufalin-20,22-dienolide; 14-dehydrobufalin;bufotalin; arenobufagin; cinobufagin; marinobufagenin; proscillaridin;scillroside; scillarenin; and 14,15-epoxy-bufalin. Without limitation,analogs and derivatives of bufalin include those that can cross theblood-brain barrier. Herein, bufadienolides and analogs and derivativesthereof are also considered bufalin analogs or derivatives thereof.Further bufalin or bufadienolide analogs and derivatives amenable to thepresent invention include those described in U.S. Pat. Nos. 3,080,362;3,136,753; 3,470,240; 3,560,487; 3,585,187; 3,639,392; 3,642,770;3,661,941; 3,682,891; 3,682,895; 3,687,944; 3,706,727; 3,726,857;3,732,203; 3,80,6502; 3,812,106; 3,838,146; 4,001,401; 4,102,884;4,175,078; 4,242,33; 4,380,624; 5,314,932; 5,874,423; and 7,087,590 andthose described in Min, et al., J. Steroid. Biochem. Mol. Biol.,91(1-2): 87-98 (2004); Kamano, Y. & Pettit, G. R. J. Org. Chem., 38(12): 2202-2204 (1973); Watabe, et al., Cell Growth Differ, 8(8): 871(1997); and Mahringer et al., Cancer Genomics and Proteomics, 7(4):191-205 (2010). Content of all of the patents and references listed inthe above paragraphs is herein incorporated by reference.

Adenosine Receptor Modulators

As used herein, the term “adenosine receptor modulator” refers to acompound that modulates at least one activity of an adenosine receptor.The term “adenosine receptor modulator” as used herein is intended toinclude agents that interact with the adenosine receptor itself, or thatcan act as an allosteric modulator of the receptor by interacting with asite on the channel complex. The term “adenosine receptor modulator” asused herein is also intended to include agents that modulate activity ofan adenosine receptor indirectly. By “indirectly,” as used in referenceto modulator interactions with adenosine receptor, means the modulatordoes not directly interact with the receptor itself, i.e., modulatorinteracts with the receptor via an intermediary. Accordingly, the term“indirectly” also encompasses the situations wherein the modulatorrequires another molecule in order to bind or interact with thereceptor.

Adenosine receptors are proteins found in animals and humans that canbind the ligand, adenosine, causing a physiological response. Adenosinereceptors have been located in a variety of tissues and cells, includinghippocampus, adipocytes, atrioventricle node, striatum, platelets,neutrophils, coronary vasculature and olfactory tubercule.

Four adenosine receptors are commonly referred to as A1, A2A, A2B, andA3. The stimulation of A1 receptors, among other things, can inhibitnerve cells, lower heart rate, slowAV nodal conduction, and promotevasoconstriction. The stimulation of A2A receptors is generallyanti-inflammatory, and can be used to sense excessive tissueinflammation, and promote coronary vasodilatation. The stimulation ofA2B generally promotes vasodilatation. The stimulation of A3 receptors,among other things, can both stimulate and inhibit cell growth, andpromote tumor growth and angiogenesis. Numerous documents describe thecurrent knowledge on adenosine receptors. These include Bioorganic &Medicinal Chemistry, 6, (1998), 619-641, Bioorganic & MedicinalChemistry, 6, (1998), 707-719, J. Med. Chem., (1998), 41, 2835-2845, J.Med. Chem., (1998), 41, 3186-3201, J. Med. Chem., (1998), 41, 2126-2133,J. Med. Chem., (1999), 42, 706-721, J. Med. Chem., (1996), 39,1164-1171, Arch. Pharm. Med. Chem., 332, 39{circumflex over ( )}1,(1999), Am. J. Physiol., 276, H1113-1116, (1999) and Naunyn Schmied,Arch. Pharmacol. 362, 375-381, (2000), content of all of which isincorporated herein by reference.

As used herein, the term “modulate” refers to a change or alternation inat least one biological activity of the adenosine receptor. Modulationcan be an increase or a decrease in activity, a change in bindingcharacteristics, or any other change in the biological, functional, orimmunological properties of the receptor. Ligands that bind to theadenosine receptor causing the inhibition of the adenosine receptorphysiological response are termed adenosine receptor antagonists.Likewise, ligands that bind to the adenosine receptor, therebygenerating a physiological response that mimics the response caused bythe adenosine receptor binding adenosine, are termed adenosine receptoragonists.

In some embodiments of the aspects described herein, the modulator is anagonist of the adenosine receptor. It will be appreciated that theadenosine receptor agonists include compounds which act both directlyand indirectly on the receptor resulting in activation of the receptor,or mimic the action of the receptor having the same net effect.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the receptor by at least 5%, at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80%, at least90%, or at least 95%, at least 98% or more relative to a control with nomodulation.

In some embodiments of the aspects described herein, at least oneactivity of the receptor is increased by at least 5%, at least 10%, atleast 15%, at least 20%, at least 25%, at least 30%, at least 40%, atleast 50%, at least 60%, at least 70%, at least 80%, at least 90%, atleast 95%, at least 98%, or 100% relative to a control with nomodulator.

Exemplary adenosine receptor modulators include, but are not limited to,2-(1-Hexynyl)-N-methyladenosine; 2-Cl-IB-MECA; 2′-MeCCPA;5′-N-ethylcarboxamido adenosine; 8-Cyclopentyl-1,3-dimethylxanthine(CPX); 8-Cyclopentyl-1,3-dipropylxinthine (DPCPX);8-Phenyl-1,3-dipropylxanthine; ATL-146e; BAY 60-6583; Caffeine; CCPA;CF-101 (IB-MECA); CGS-21680; CP-532,903; CVT-6883; GR 79236;istradefylline; LUF-5835; LUF-5845; MRE3008F20; MRS-1191; MRS-1220;MRS-1334; MRS-1523; MRS-1706; MRS-1754; MRS-3558; MRS-3777;N6-Cyclopentyladenosine; PSB 36; PSB-0788; PSB-10; PSB-11; PSB-1115;PSB-603; Regadenoson; SCH-442,416; SCH-58261; SDZ WAG 994; theophylline;VUF-5574; ZM-241,385; and the like.

Exemplary agonists of adenosine receptor agonists include, but are notlimited to, GR 79236; SDZ WAG 994; ATL-146e; CGS-21680; Regadenoson;5′-N-ethylcarboxamidoadenosine; BAY 60-6583; LUF-5835; LUF-5845;2-(1-Hexynyl)-N-methyladenosine; CF-101 (IB-MECA); 2-Cl-IB-MECA;CP-532,903; MRS-3558; N6-cyclopentyladenosine (CPA), N-ethylcarboxamidoadenosine (NECA),2-[p-(2-carboxyethyl)phenethyl-amino-5′-N-ethylcarboxamido adenosine(CGS-21680); 2-chloroadenosine; N6-[2-(3,5-demethoxyphenyl)-2-(2-methoxyphenyl]ethyladenosine;2-chloro-N6-cyclopentyladenosine (CCPA); 2′-MeCCPA;N-(4-aminobenzyl)-9-[5-(mefhylcarbonyl)-beta-D-robofuranosyl]adenine(AB-MECA);([IS-[1a,2b,3b,4a(S*)]]-4-[7-[[2-(3chloro-2-thienyl)-1-methyl-propyl]amino]-3H-imidazole[4, 5-b]pyridyl-3-yl]cyclopentane carboxamide(AMP579); N6-(R)-phenylisopropyladenosine (R-PLA);aminophenylethyladenosine 9APNEA) and cyclohexyladenosine (CHA);N-[3-(R)-tetrahydrofuranyl]-6-aminopurine riboside (CVT510); CVT-2759;allosteric enhancers such as PD81723;N6-cyclopentyl-2-(3-phenylaminocarbonyltriazene-1-yl)adenosine (TCPA);2-amino-3-naphthoylthiophenes78; and the like.

In some embodiments, the adenosine receptor agonist can beN6-cyclopentyladenosine

Gamma-Secretase Ligands

As used herein, the term “modulate,” with reference to thegamma-secretase means to regulate positively or negatively the normalfunctioning of a gamma-secretase. Thus, the term modulate can be used torefer to an increase, decrease, masking, altering, overriding orrestoring the normal functioning of a gamma-secretase. A gamma-secretasemodulator can be a gamma-secretase agonist or a gamma-secretaseantagonist.

In some embodiments of the aspects described herein, the modulatormodulates at least one activity of the gamma-secretase by at least 5%,at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, atleast 40%, at least 50%, at least 60%, at least 70%, at least 80%, atleast 90%, or at least 95%, at least 98% or more relative to a controlwith no modulation.

As used herein, the terms “gamma-secretase protein” and“gamma-secretase” refer to a protein that exhibits gamma-secretaseactivity which includes: recognizing a polypeptide substrate having agamma-secretase cleavage sequence; and catalyzing cleavage of thegamma-secretase cleavage sequence, at the gamma-secretase cleavage site,to produce substrate cleavage products.

Gamma-secretase is a macromolecular proteolytic complex composed of atleast four proteins: presenilin (PS), nicastrin (NCT), PEN-2 and APH-1(De Strooper, 2003, Neuron 38:9-12). Recently, CD147 and TMP21 have beenfound to be associated with the gamma-secretase complex (Chen, et al.,2006, Nature 44011208-1212; Zhou et al., 2005, Proc. Natl. Acad. Sci.USA, 102:7499-7504). Among these known components, PS is believed tocontain the active site of gamma-secretase, recognized as an aspartylprotease (Esler et al., 2000, Nat. Cell. Biol., 2:428:434; Li et al.,2000, Nature 4051689-694; Wolfe et al., 1999, Nature 3981513-517).Considerable effort has been made to understand the process ofgamma-secretase substrate recognition and its catalytic machinery. APS-dependent protease can process any singlepass transmembrane (TM)protein regardless of its primary sequence as long as the TM proteinextracellular domain is smaller than 300 amino acids. Moreover, the sizeof the extracellular domain appears to determine the efficiency ofsubstrate cleavage (Struhl and Adachi, 2000, Mol. Cell 6:625636).

Exemplary gamma-secretase inhibitors include, but are not limited to,those described in U.S. Patent Application Publication Nos.US2003/0216380; US2006/0009467; US2004/0048848; US2004/0171614;US2005/0085506; US2006/0100427; US2005/0261495; US2007/0299053;US2006/0264417; US2006/0258638; US2005/0245501; US2003/0134841;US2008/004,5533; US2007/0213329; US2006/0041020; US2004/016404; andUS2003/0114496, U.S. Pat. Nos. 7,122,675; 6,683,091; 7,208,602; 7,256,186; 6,967,196; 7,304,056; 7,304,055; 7,101,870; 6,962,913; 6,794,381;7,304,094; and 6,984,663, and PCT Publication Nos. WO03/013527;WO03/066592; WO00/247671; WO00/050391; WO00/007995; and WO03/018543,content of all of which is incorporated herein by reference. Additionalexemplary gamma secretase modulators include certain nonsteroidalanti-inflammatory drugs (NSAIDs) and their analogs as described in U.S.Patent Publication No. US 2002/0128319, PCT Publication No. WO01/78721,and Weggen et al., Nature, 414 (2001) 212-16; Morihara et al., J.Neurochem., 83 (2002), 1009-12; and Takahashi et al., J. Biol. Chem.,278 (2003), 18644-70), content of all of which is incorporated herein byreference.

In some embodiments, the gamma-secretase modulator inhibits binding of asubstrate by at least about or about any one of 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 95% or 100% relative to a control. Binding of asubstrate to a gamma-secretase can be determined by any method known toone of skill in the art.

In some embodiments, the gamma-secretase modulator reduces an activityof a gamma-secretase by at least 5%, at least 10%, at least 20%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 95%, at least 98%, or 100% (e.g.complete loss of activity) relative to an uninhibited control.

In some embodiments, the gamma-secretase modulator enhances an activityof a gamma-secretase by at least 5%, 10%, 20%, 30%, 40%, 50%, 50%, 70%,80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold ormore relative to an unactivated control.

In some embodiments, the gamma-secretase modulator is capable of bindingto the active site of a GPCR (e.g., a binding site for a substrate).

In some embodiments, the serotonin receptor modulator is capable ofbinding to an allosteric site of a gamma-secretase.

In some embodiments of this and other aspects described herein, the GPCRantagonist has an IC50 of less than or equal to 500 nM, less than orequal to 250 nM, less than or equal to 100 nM, less than or equal to 50nM, less than or equal to 10 nM, less than or equal to 1 nM, less thanor equal to 0.1 nM, less than or equal to 0.01 nM, or less than or equalto 0.001 nM.

In some embodiments of this and other aspects of the invention, the GPCRagonist has an EC50 of less than or equal to 500 nM, 250 nM, 100 nM, 50nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM or 0.001 nM.

Corticosteroids

As used herein, the term “corticosteroid” refers to a class of steroidhormones that are produced in the adrenal cortex or producedsynthetically. Corticosteroids are involved in a wide range ofphysiologic systems such as stress response, immune response andregulation of inflammation, carbohydrate metabolism, protein catabolism,blood electrolyte levels, and behavior. Corticosteroids are generallygrouped into four classes, based on chemical structure. Group Acorticosteroids (short to medium acting glucocorticoids) includehydrocortisone, hydrocortisone acetate, cortisone acetate, tixocortolpivalate, prednisolone, methylprednisolone, and prednisone. Group Bcorticosteroids include triamcinolone acetonide, triamcinolone alcohol,mometasone, amcinonide, budesonide, desonide, fluocinonide, fluocinoloneacetonide, and halcinonide. Group C corticosteroids includebetamethasone, betamethasone sodium phosphate, dexamethasone,dexamethasone sodium phosphate, and fluocortolone. Group Dcorticosteroids include hydrocortisone-17-butyrate,hydrocortisone-17-valerate, aclometasone dipropionate, betamethasonevalerate, betamethasone dipropionate, prednicarbate,clobetasone-17-butyrate, clobetasol-17-propionate, fluocortolonecaproate, fluocortolone pivalate, and fluprednidene acetate.

Without limitations, a corticosteroid can be selected from the groupconsisting of small or large organic or inorganic molecules;monosaccharides; disaccharides; trisaccharides; oligosaccharides;polysaccharides; biological macromolecules, e.g., proteins, peptides,peptide analogs and derivatives thereof, peptidomimetics, nucleic acids,nucleic acid analogs and derivatives, enzymes, antibodies, portion orfragments of antibodies; an extract made from biological materials suchas bacteria, plants, fungi, or animal cells or tissues; naturallyoccurring or synthetic compositions; and any combinations thereof.

Exemplary corticosteroids include, but are not limited to, aldosternone,beclomethasone, beclomethasone dipropionate, betametahasone,betametahasone-21-phosphate disodium, betametahasone valerate,budesonide (also referred to as Bud herein), clobetasol, clobetasolpropionate, clobetasone butyrate, clocortolone pivalate, cortisol,cortisteron, cortisone, dexamethasone, dexamethasone acetate,dexamethasone sodium phosphate, diflorasone diacetate, flucinonide,fludrocortisones acetate, flumethasone, flunisolide, flucionoloneacetonide, fluticasone furate, fluticasone propionate, halcinonide,halpmetasone, hydrocortisone, hydroconrtisone acetate, hydrocortisonesuccinate, 16α-hydroxyprednisolone, isoflupredone acetate, medrysone,methylprednisolone, prednacinolone, predricarbate, prednisolone,prednisolone acetate, prednisolone sodium succinate, prednisone,triamcinolone, triamcinolone, and triamcinolone diacetate.

As used herein, the term corticosteroid is intended to include thefollowing generic and brand name corticosteroids: cortisone (CORTONEACETATE, ADRESON, ALTESONA, CORTELAN, CORTISTAB, CORTISYL, CORTOGEN,CORTONE, SCHEROSON); dexamethasone-oral (DECADRON-ORAL, DEXAMETH,DEXONE, HEXADROL-ORAL, DEXAMETHASONE INTENSOL, DEXONE 0.5, DEXONE 0.75,DEXONE 1.5, DEXONE 4); hydrocortisone-oral (CORTEF, HYDROCORTONE);hydrocortisone cypionate (CORTEF ORAL SUSPENSION);methylprednisolone-oral (MEDROL-ORAL); prednisolone-oral (PRELONE,DELTA-CORTEF, PEDIAPRED, ADNISOLONE, CORTALONE, DELTACORTRIL,DELTASOLONE, DELTASTAB, DIADRESON F, ENCORTOLONE, HYDROCORTANCYL,MEDISOLONE, METICORTELONE, OPREDSONE, PANAAFCORTELONE, PRECORTISYL,PRENISOLONA, SCHERISOLONA, SCHERISOLONE); prednisone (DELTASONE, LIQUIDPRED, METICORTEN, ORASONE 1, ORASONE 5, ORASONE 10, ORASONE 20, ORASONE50, PREDNICEN-M, PREDNISONE INTENSOL, STERAPRED, STERAPRED DS, ADASONE,CARTANCYL, COLISONE, CORDROL, CORTAN, DACORTIN, DECORTIN, DECORTISYL,DELCORTIN, DELLACORT, DELTADOME, DELTACORTENE, DELTISONA, DIADRESON,ECONOSONE, ENCORTON, FERNISONE, NISONA, NOVOPREDNISONE, PANAFCORT,PANASOL, PARACORT, PARMENISON, PEHACORT, PREDELTIN, PREDNICORT,PREDNICOT, PREDNIDIB, PREDNIMENT, RECTODELT, ULTRACORTEN, WINPRED);triamcinoloneoral (KENACORT, ARISTOCORT, ATOLONE, SHOLOG A, TRAMACORT-D,TRI-MED, TRIAMCOT, TRISTOPLEX, TRYLONE D, U-TRI-LONE).

Other exemplary corticosteroid drugs include cortisone, Cortisol,hydrocortisone (1103, 17-dihydroxy, 21-(phosphonooxy)-pregn-4-ene,3,20-dione disodium), dihydroxycortisone, dexamethasone(21-(acetyloxy)-9fluoro-β,17-dihydroxy-16α-methylpregna-1,4-diene-3,20-dione),and highly derivatized steroid drugs such as beconase (beclomethasonedipropionate, which is 9-chloro11(3,17,21,trihydroxy-16β-methylpregna-1,4diene-3,20-dione 17,21-dipropionate).Other examples of corticosteroids include flunisolide, prednisone,prednisolone, methylprednisolone, triamcinolone, deflazacort andbetamethasone.

In some embodiments, the corticosteroid can be Budesonide

Synergistic Effect

The inventors have also discovered that many of the hit compounds have asynergistic effect on satellite cell proliferation when given togetherwith a growth factor. Accordingly, in some embodiments of the aspectsdescribed herein, a compound described herein is contacted with thesatellite cell along with a growth factor. Exemplary growth factorsinclude, but are not limited to, basic epidermal growth factor (bEGF),fibroblast growth factors (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosins,platelet-derived growth factors (PDGF), insulin binding growth factors(IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growthfactor (TGF), TGF-alpha, TGF-beta, cartilage inducing factors-A and —B,osteoid-inducing factors, osteogenin, bone morphogenic proteins, andother bone growth factors, collagen growth factors, heparin-bindinggrowth factor-1 or -2, and their biologically active derivatives.

The term “synergistic” as used herein is defined to mean a combinationof components wherein the activity of the combination is greater thanthe additive of the individual activities of each component of thecombination. In some embodiments, the activity of the combination is atleast 5%, at least 10%, at least 15%, at least 20%, at least 25%, atleast 30%, at least 40%, at least 50%, at least 60%, at least 70%, atleast 80%, at least 90%, at least 1-fold, at least 2-fold, at least3-fold, at least 4-fold, at least 5-fold, at least 10-fold, at least50-fold, at least 100-fold or greater than the additive of theindividual activities of each component of the combination.

The compound and the growth factor can be contacted with the satellitecell in any ratio. The ratio can be a mole:mole ratio or a weight:weightratio For example, the ratio can range from 100:1 to 1:100. In someembodiment, proliferation enhancer and the growth factor are in a ratiofrom 20:1 to 1:20. In some embodiments, the proliferation enhancer andthe growth factor are in a ratio from 10:1 to 1:10. In some embodiments,the proliferation enhancer and the growth factor are in a ratio from 5:1to 1:5. In some embodiments, the proliferation enhancer and the growthfactor are in a ratio from 15:1 to 1:5. In some embodiments, theproliferation enhancer and the growth factor are in a ratio from 10:1 to1:1. In one embodiment, proliferation enhancer and the growth factor areused in a 1:1 ratio.

In some embodiments, the growth factor can be selected from the groupconsisting of basic epidermal growth factor (bEGF), fibroblast growthfactors (FGF), FGF-1, FGF-2 (bFGF), FGF-4, thymosins, platelet-derivedgrowth factors (PDGF), insulin binding growth factors (IGF), IGF-1,IGF-2, epidermal growth factor (EGF), transforming growth factor (TGF),TGF-alpha, TGF-beta, cartilage inducing factors-A and —B,osteoid-inducing factors, osteogenin, bone morphogenic proteins, otherbone growth factors, collagen growth factors, heparin-binding growthfactor-1 or -2, and their biologically active derivatives.

Synergistic Compositions

As discussed herein, the inventors have discovered, inter alia, thatsome of the proliferation enhancers show a synergistic effect onsatellite cell proliferation when used together with a growth factor.Accordingly, the disclosure also provided synergistic compositionscomprising a proliferation enhancer and a growth factor. The growthfactor can be selected

Without limitations, the proliferation enhancer and the growth factorcan be present in any ratio in the synergistic composition, and theratio can mole:mole or weight:weight. For example, the proliferationenhancer and the growth factor can be in a ratio from 100:1 to 1:100. Insome embodiment, proliferation enhancer and the growth factor are in aratio from 20:1 to 1:20. In some embodiments, the proliferation enhancerand the growth factor are in a ratio from 10:1 to 1:10. In someembodiments, the proliferation enhancer and the growth factor are in aratio from 5:1 to 1:5. In some embodiments, the proliferation enhancerand the growth factor are in a ratio from 15:1 to 1:5. In someembodiments, the proliferation enhancer and the growth factor are in aratio from 10:1 to 1:1. In one embodiment, proliferation enhancer andthe growth factor are used in a 1:1 ratio. In some embodiments, theproliferation enhancer and the growth factor can in a ratio of 50:1,40:1, 30:1, 25:1, 20:1, 15:1, 10:1, 5:1, 3:1, 2:1, 1:1.75, 1.5:1, or1.25:1 to 1:1.25, 1:1.5, 1.75, 1:2, 1:3, 1:4, 1:5, 1:10, 1:15, 1:20,1:20, 1:30, 1:40, or 1:50.

Contacting of Satellite Cells with Compounds

The satellite cell population can be contacted with the proliferationenhancer in a cell culture e.g., in vitro or ex vivo, or theproliferation enhancer can be administrated to a subject, e.g., in vivo.In some embodiments of the invention, a proliferation enhancer describedherein can be administrated to a subject for repairing or regenerating adamaged muscle tissue.

The term “contacting” or “contact” as used herein in connection withcontacting a population of satellite cells includes subjecting thesatellite cells to an appropriate culture media which comprises theindicated compound or agent. Where the satellite cell population is invivo, “contacting” or “contact” includes administering the proliferationenhancer or agent in a pharmaceutical composition to a subject via anappropriate administration route such that the proliferation enhancer oragent contacts the satellite cell population in vivo.

For in vivo methods, a therapeutically effective amount of a compounddescribed herein can be administered to a subject. Methods ofadministering compounds to a subject are known in the art and easilyavailable to one of skill in the art. Promoting satellite cellproliferation in a subject can lead to treatment, prevention oramelioration of a number of diseases, disorders or conditions which arecaused by a damaged muscle tissue.

Satellite cells suitable for use in ex vivo methods can be obtained fromsubject according to methods well known to those skilled in the art.

The term “ex vivo” refers to cells which are removed from a livingorganism and cultured outside the organism (e.g., in a test tube). Forex vivo methods, satellite cells can include autologous satellite cells,i.e., a cell or cells taken from a subject who is in need of treatmentfor muscle damage or repair. Autologus satellite cells have theadvantage of avoiding any immunologically-based rejection of the cells.Alternatively, the cells can be heterologous, e.g., taken from a donor.The second subject can be of the same or different species. Typically,when the cells come from a donor, they will be from a donor who issufficiently immunologically compatible with the recipient, i.e., willnot be subject to transplant rejection, to lessen or remove the need forimmunosuppression. In some embodiments, the cells are taken from axenogeneic source, i.e., a non-human mammal that has been geneticallyengineered to be sufficiently immunologically compatible with therecipient, or the recipient's species. Methods for determiningimmunological compatibility are known in the art, and include tissuetyping to assess donor-recipient compatibility for HLA and ABOdeterminants. See, e.g., Transplantation Immunology, Bach andAuchincloss, Eds. (Wiley, John & Sons, Incorporated 1994).

Without wishing to be bound by theory any suitable cell culture mediacan be used for ex vivo methods of the invention. For example, inventorshave discovered that the cells can survive in minimum of 10% serummedium. While the cells can survive in suspension culture with at least30% serum, they may need to be adherent when medium with 10% serum isused in absence of bFGF. Cells are optimal in cultures when they areadherent on laminin. After ex vivo contact with a compound describedherein, when the satellite cells have reached a desired populationnumber or density, e.g., about 1×10⁶, 2×10⁶, 3×10⁶, 4×10⁶, 5×10⁶, 6×10⁶,7×10⁶, 8×10⁶, 9×10⁶, 1×10⁷, 2×10⁷, or more cells, the cells can betransplanted in a subject who is in need of treatment for muscle repairor damage. The cells can be transplanted in a subject from whom thecells were originally obtained or in different subject.

When the satellite cells are contacted with a proliferation enhancer,the proliferation enhancer can have a direct or an indirect affect onthe satellite cells. As used herein, a “direct affect” means that theproliferation enhancer is directly interacting with the satellite cells,e.g., binding to a cell surface receptor on the satellite cell, taken upinto the satellite cells. As used herein, an “indirect affect” meansthat the proliferation enhancer does not directly interacts with thesatellite cell. For example, the proliferation enhancer can interactwith a non-satellite cell and indirectly influence the proliferation ofsatellite cell. Without wishing to be bound by theory, the proliferationenhancer can indirectly influence a satellite cell by inducingexpression and/or secretion of a molecule from a non-satellite cell, andthis molecule then directly or indirectly influencing the proliferationof satellite cells.

As used herein, the term “damaged muscle tissue” refers to a muscletissue, such as a skeletal or cardiac muscle that has been altered forinstance by a physical injury or accident, disease, infection, over-use,loss of blood circulation, or by genetic or environmental factors. Adamaged muscle tissue can be a dystrophic muscle or an ageing muscle.Exemplary symptoms of muscle damage include, but are not limited to,swelling, bruising or redness, open cuts as a consequence of an injury,pain at rest, pain when specific muscle or the joint in relation to thatmuscle is used, weakness of the muscle or tendons, and an inability touse the muscle at all.

In some embodiments of this and other aspects described herein, thedamaged muscle tissue results from muscle atrophy/wasting.

In some embodiments of this and other aspects described herein, thedamaged muscle tissue results from sarcopenia. As used herein, the term“sarcopenia” refers to the loss of muscle mass and function thatinevitably occurs with aging. Sarcopenia is responsible for decreasedlevels of physical activity which, in turn, can result in increased bodyfat and a further loss of muscle. Loss of muscle mass results from anegative net balance between muscle protein synthesis and muscle proteinbreakdown. The etiology of this loss of skeletal muscle mass andfunction is not believed to be clear. Reduced levels of physicalactivity, loss of motor units secondary to changes in the centralnervous system, and inadequate protein intake have all been implicated

In some embodiments of this and other aspects described herein, thedamaged muscle tissue results from a physical injury.

In some embodiments of this and other aspects of the invention, thedamaged muscle is skeletal muscle.

In some embodiments, the subject has or is otherwise affected by muscleinjury, insult or disease.

In some embodiments of this and other aspects described herein, diseaseresulting in damaged muscle tissue is a myopathy. Without limitation,myopathy can be a congenital myopathy or an acquired myopathy. Exemplarymyopathies include, but are not limited to, dystrophies, myotonia(neuromytonia), congenital myopathies (e.g., nemaline myopathy,multi/minicore myopathy, centronuclear myopathy (or myotubularmyopathy)), mitochondrial myopathies, familial periodic paralysis,inflammatory myopathies, metabolic myopathies (e.g., glycogen storagedisease and lipid storage disorder), dermatomyositis, polymyositisinclusion body myositis, myositis ossificans, rhabdomyolysis andmyoglobinuirias.

In some embodiments of this and other aspects described herein, myopathyis a dystrophy selected from the group consisting of muscular dystrophy,Duchenne muscular dystrophy, Becker's muscular dystrophy, Reflexsympathetic dystrophy, Retinal dystrophy, Conal dystrophy, Myotonicdystrophy, Corneal dystrophy, and any combinations thereof.

Congenital myopathy is a term sometimes applied to hundreds of distinctneuromuscular disorders that may be present at birth, but it is usuallyreserved for a group of rare inherited primary muscle disorders thatcause hypotonia and weakness at birth or during the neonatal period and,in some cases, delayed motor development later in childhood. Patientssuffer from weakness ranging from mild (late childhood onset and abilityto walk through adulthood) to severe (respiratory insufficiency anddeath within the first year of life).

The most common types of congenital myopathy are nemaline myopathy,myotubular myopathy, central core myopathy, congenital fiber typedisproportion, and multicore myopathy. They are distinguished primarilyby their histological features, symptoms, and prognosis. Diagnosis isindicated by characteristic clinical findings and confirmed by musclebiopsy.

In certain embodiments, a therapeutically effective amount of a compounddescribed herein can be administered to a subject to treat any diseasein which small muscles (e.g., sphincter muscles) are affected. Incertain embodiments, a therapeutically effective amount of a compounddescribed herein can be administered to a subject to treat esophagealdiseases (e.g., esophageal reflux disease and other diseases resultingfrom the loss of esophageal muscle control, tone or motility). Incertain embodiments, a therapeutically effective amount of a compounddescribed herein can be administered to a subject to treat urinaryincontinence. In certain embodiments, a therapeutically effective amountof a compounds described herein can be administered to a subject totreat fecal incontinence. In certain aspects, a therapeuticallyeffective amount of a compound contemplated herein may be administeredto a subject to increase or improve sphincter muscle tone.

X-Linked Myotubular myopathy (XLMTM): Myotubular myopathy is autosomalor X-linked. The more common autosomal variation produces mild weaknessand hypotonia in both sexes. The X-linked variation affects males andresults in severe skeletal muscle weakness and hypotonia, facialweakness, impaired swallowing, and respiratory muscle weakness andrespiratory failure. However, female carriers rarely express significantclinical symptoms. Most patients die within the first year of life fromrespiratory failure. Some patients survive for several years and mayshow spontaneous improvement of respiratory function after birth. XLMTMhas also been referred to as CNM, MTMX, X-linked centronuclear myopathy,and XMTM in the art.

The characteristic muscle histopathology consists of small roundedmuscle fibers with centrally located nuclei surrounded by a halo devoidof contractile elements but containing mitochondria. The MTM1 gene ismutated in vast majority of XLMTM patients. This gene is ubiquitouslyexpressed and shows a muscle-specific alternative transcript due to theuse of a different polyadenylation signal. Over one hundred thirty-threedifferent disease associated mutations have been described in the MTM1gene. A list of MTM1 mutations is maintained on the web at the HumanGene Mutation Database under entry for XLMTM and can be accessed at thefollowing address: uwcm.ac.uk/uwcm/mg/search/119439. Mutations in theMTM1 gene are widespread throughout the gene, although more have beenfound in exon 4, 12, 3, 8, 9, and 11, in that order when comparing thenumber of mutations to the nucleotide-length ratio for each exon. For areview of MTM1 mutations in X-Linked Myotubular Myopathy see J. Laporteet al. 2000, 15:393-409.

Mutations in the MTM1 gene include missense, nonsense, small insertionsor deletions, large deletions and splice-site mutations. Most pointmutations are truncating; however about 25% of the mutations aremissense. While most truncating and splice mutations are associated witha severe phenotype, some missense mutations are associated with milderor less severe phenotype and prolonged survival.

Nemaline myopathy: Nemaline myopathy can be autosomal dominant orrecessive and result from various mutations on different chromosomes.Nemaline myopathy may be severe, moderate, or mild in neonates. Severelyaffected patients may experience weakness of respiratory muscles andrespiratory failure. Moderate disease produces progressive weakness inmuscles of the face, neck, trunk, and feet, but life expectancy may benearly normal. Mild disease is nonprogressive, and life expectancy isnormal.

Central core myopathy: Inheritance is autosomal dominant. Most affectedpatients develop hypotonia and mild proximal muscle weakness asneonates. Many also have facial weakness. Weakness is nonprogressive,and life expectancy is normal. However, patients are at higher risk ofdeveloping malignant hyperthermia (the gene associated with central coremyopathy is also associated with increased susceptibility to malignanthyperthermia).

Congenital fiber type disproportion: Congenital fiber type disproportionis inherited, but the pattern is poorly understood. Hypotonia andweakness of the face, neck, trunk, and limbs are often accompanied byskeletal abnormalities and dysmorphic features. Most affected childrenimprove with age, but a small percentage develops respiratory failure.

Multicore myopathy: Multicore myopathy is usually autosomal recessivebut may be autosomal dominant. Infants typically present with proximalweakness, but some children present later with generalized weakness.Progression is highly variable.

In some embodiments, a method described herein also comprises the stepof diagnosing a subject for congenital myopathy before onset ofadministering a compound described herein. A subject can be diagnosedfor congenital myopathy based on the symptoms presented by the subject.

Generally, symptoms of congenital myopathies include, but are notlimited to, depressed reflexes, enlarged muscles, difficulty relaxingmuscles following contractions, stiff muscles, and rigid muscles.Specific symptoms are described below.

Central core disease is characterized by a mild, non-progressive muscleweakness. Signs of central core disease usually appear in infancy orearly childhood and may present even earlier. There may be decreasedfetal movements and breech (feet first) presentation in utero. The mainfeatures of CCD are poor muscle tone (hypotonia), muscle weakness, andskeletal problems including congenital hip dislocation, scoliosis(curvature of the spine), pes cavus (high-arched feet), and clubbedfeet. Children with CCD experience delays in reaching motor milestonesand tend to sit and walk much later than those without the disorder. Achild with the disease usually cannot run easily, and may find thatjumping and other physical activities are often impossible. Althoughcentral core disease may be disabling, it usually does not affectintelligence or life expectancy.

People who have central core disease are sometimes vulnerable tomalignant hyperthermia (MH), a condition triggered by anesthesia duringsurgery. MH causes a rapid, and sometimes fatal, rise in bodytemperature, producing muscle stiffness.

There is variability in age of onset, presence of symptoms, and severityof symptoms in nemaline myopathy (NM). Most commonly, NM presents ininfancy or early childhood with weakness and poor muscle tone. In somecases there may have been pregnancy complications such as polyhydramnios(excess amniotic fluid) and decreased fetal movements. Affected childrenwith NM tend to have delays in motor milestones such as rolling over,sitting and walking. Muscle weakness commonly occurs in the face, neckand upper limbs. Over time, a characteristic myopathic face (a long facethat lacks expression) develops. Skeletal problems including chestdeformities, scoliosis, and foot deformities may develop. In the mostsevere cases of NM, feeding difficulties and potentially fatalrespiratory problems may also occur. In those who survive the first twoyears of life, muscle weakness tends to progress slowly or not at all.

Typically the X-linked form of MTM (XLMTM) is the most severe of thethree forms (X-linked, autosomal recessive, and autosomal dominant).XLMTM usually presents as a newborn male with poor muscle tone andrespiratory distress. The pregnancy may have been complicated bypolyhydramnios and decreased fetal movements. Of those who survive thenewborn period, many will at least partially depend on a ventilator forbreathing. Because of the risk of aspiration, many will also have agastrostomy tube (G-tube). Boys with XLMTM can experience significantdelays in achieving motor milestones and may not ever walkindependently. They tend to be tall with a characteristic facialappearance (long, narrow face with a highly arched roof of the mouth andcrowded teeth). Intelligence is generally not affected. Medicalcomplications that may develop include: scoliosis, eye problems (eyemuscle paralysis and droopy eyelids), and dental malocclusion (severecrowding). In XLMTM, other problems including undescended testicles,spherocytosis, peliosis, elevated liver enzymes, and gallstones mayoccur.

The autosomal recessive and autosomal dominant forms of MTM tend to havea milder course than the X-linked form. The autosomal recessive form canpresent in infancy, childhood, or early adulthood. Common featuresinclude generalized muscle weakness with or without facial weakness andophthalmoplegia (paralysis of the eye muscles). Although feeding andbreathing problems can occur, affected individuals usually surviveinfancy. Onset of the autosomal dominant form ranges from late childhoodthrough early adulthood. It tends to be the mildest of the three formsof MTM. Unlike the X-linked form of the condition, problems with otherorgans (such as the liver, kidneys, and gall bladder) haven't beenreported with the autosomal recessive and autosomal dominant forms ofMTM.

Diagnosis of a congenital myopathy generally includes evaluation of thesubject's personal and family history, physical and neurologicalexaminations that test reflexes and strength, and specialized tests.Since there is overlap between the symptoms of a congenital myopathy andother neuromuscular disorders, a number of tests may be performed tohelp narrow down the diagnosis. Serum CK (creatinine kinase) analysis,EMG (electromyelogram), nerve conduction studies, and muscle ultrasoundtend to be of limited value in making this diagnosis. The definitivediagnosis of a congenital myopathy usually relies upon genetic testingand/or muscle biopsy. Also, muscle biopsy can be used to determine apatient's susceptibility to malignant hyperthermia.

X-linked myotubular myopathy: Diagnosis of X-linked MTM is usually madeon muscle biopsy. Findings include: centrally located nuclei in musclefibers that look like myotubules, absence of structures known asmyofibrils, and possibly, persistence of certain proteins usually seenin fetal muscle cells. Gene testing detects a mutation (disease-causinggene change) in up to 97-98% of people with the X-linked form. Genetesting can comprise: (i) complete gene sequencing of the MTM1 gene;(ii) mutation screening (scanning) by methods such as single-strandedconformational polymorphism (SSCP) or denaturing gradienthigh-performance liquid chromatography (DHPLC), followed by sequencingof the abnormal fragments; and (iii) deletion testing. XLMTM can also bediagnosed by measuring levels of myoubilarin. Patients with known MTM1mutations usually show abnormal myotubularin levels.

Central core disease: The muscle biopsy from a person with CCD typicallydisplays a metabolically inactive “core” or central region that appearsblank when stained (tested) for certain metabolic enzymes (proteins)that should be there. These central regions also lack mitochondria, theenergy producing “factories” of the cells. Genetic testing for RYR1mutations is available on a research basis. The same genetic test may beused to determine the presence of the gene change in family members whomay have or be at-risk for the disease. For families in which a RYR1mutation has been found, prenatal diagnosis may be possible using theDNA of fetal cells obtained from chorionic villus sampling (CVS) oramniocentesis.

Nemaline myopathy: The clinical diagnosis of NM is suspected in aninfant under age one with muscle weakness and hypotonia (decreasedmuscle tone). Definitive diagnosis of nemaline myopathy is made bydemonstration of nemaline bodies, rod-shaped structures characteristicof this disease, using a specific stain known as “Gomori trichrome” on amuscle biopsy sample. Muscle biopsy may also show predominance ofstructures known as type I fibers. Genetic testing is available on aclinical basis for one gene, the ACTA1 gene located on the long arm ofchromosome 1. About 15% of NM cases are due to mutations in this gene.Prenatal diagnosis is possible for families with known ACTA1 mutations.The DNA of a fetus can be tested using cells obtained from chorionicvillus sampling (CVS) or amniocentesis.

Pharmaceutical Compositions

For administration to a subject, the proliferation enhancers can beprovided in pharmaceutically acceptable compositions. Thesepharmaceutically acceptable compositions comprise atherapeutically-effective amount of one or more of the proliferationenhancers, formulated together with one or more pharmaceuticallyacceptable carriers (additives) and/or diluents. As described in detailbelow, the pharmaceutical compositions of the present invention can bespecially formulated for administration in solid or liquid form,including those adapted for the following: (1) oral administration, forexample, drenches (aqueous or non-aqueous solutions or suspensions),gavages, lozenges, dragees, capsules, pills, tablets (e.g., thosetargeted for buccal, sublingual, and systemic absorption), boluses,powders, granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; (3) topical application, for example,as a cream, ointment, or a controlled-release patch or spray applied tothe skin; (4) intravaginally or intrarectally, for example, as apessary, cream or foam; (5) sublingually; (6) ocularly; (7)transdermally; (8) transmucosally; or (9) nasally. Additionally,compounds can be implanted into a patient or injected using a drugdelivery system. See, for example, Urquhart, et al., Ann. Rev.Pharmacol. Toxicol. 24: 199-236 (1984); Lewis, ed. “Controlled Releaseof Pesticides and Pharmaceuticals” (Plenum Press, New York, 1981); U.S.Pat. No. 3,773,919; and U.S. Pat. No. 35 3,270,960, content of all ofwhich is herein incorporated by reference.

As used here, the term “pharmaceutically acceptable” refers to thosecompounds, materials, compositions, and/or dosage forms which are,within the scope of sound medical judgment, suitable for use in contactwith the tissues of human beings and animals without excessive toxicity,irritation, allergic response, or other problem or complication,commensurate with a reasonable benefit/risk ratio.

As used here, the term “pharmaceutically-acceptable carrier” means apharmaceutically-acceptable material, composition or vehicle, such as aliquid or solid filler, diluent, excipient, manufacturing aid (e.g.,lubricant, talc magnesium, calcium or zinc stearate, or steric acid), orsolvent encapsulating material, involved in carrying or transporting thesubject compound from one organ, or portion of the body, to anotherorgan, or portion of the body. Each carrier must be “acceptable” in thesense of being compatible with the other ingredients of the formulationand not injurious to the patient. Some examples of materials which canserve as pharmaceutically-acceptable carriers include: (1) sugars, suchas lactose, glucose and sucrose; (2) starches, such as corn starch andpotato starch; (3) cellulose, and its derivatives, such as sodiumcarboxymethyl cellulose, methylcellulose, ethyl cellulose,microcrystalline cellulose and cellulose acetate; (4) powderedtragacanth; (5) malt; (6) gelatin; (7) lubricating agents, such asmagnesium stearate, sodium lauryl sulfate and talc; (8) excipients, suchas cocoa butter and suppository waxes; (9) oils, such as peanut oil,cottonseed oil, safflower oil, sesame oil, olive oil, corn oil andsoybean oil; (10) glycols, such as propylene glycol; (11) polyols, suchas glycerin, sorbitol, mannitol and polyethylene glycol (PEG); (12)esters, such as ethyl oleate and ethyl laurate; (13) agar; (14)buffering agents, such as magnesium hydroxide and aluminum hydroxide;(15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18)Ringer's solution; (19) ethyl alcohol; (20) pH buffered solutions; (21)polyesters, polycarbonates and/or polyanhydrides; (22) bulking agents,such as polypeptides and amino acids (23) serum component, such as serumalbumin, HDL and LDL; (22) C₂-C₁₂ alcohols, such as ethanol; and (23)other non-toxic compatible substances employed in pharmaceuticalformulations. Wetting agents, coloring agents, release agents, coatingagents, sweetening agents, flavoring agents, perfuming agents,preservative and antioxidants can also be present in the formulation.The terms such as “excipient”, “carrier”, “pharmaceutically acceptablecarrier” or the like are used interchangeably herein.

The phrase “therapeutically-effective amount” as used herein means thatamount of a compound, material, or composition comprising a compounddescribed herein which is effective for producing some desiredtherapeutic effect in at least a sub-population of cells in an animal ata reasonable benefit/risk ratio applicable to any medical treatment. Forexample, an amount of a compound administered to a subject that issufficient to produce a statistically significant, measurable satellitecell proliferation or muscle repair or regeneration.

As used herein, the term “repair” refers to a process by which thedamages of a muscle tissue are alleviated or completely eliminated. Insome embodiments, at least one symptom of muscle tissue damage isalleviated by at least 5%, at least 10%, at least 20%, at least 30%, atleast 40%, or at least 50%.

Determination of a therapeutically effective amount is well within thecapability of those skilled in the art. Generally, a therapeuticallyeffective amount can vary with the subject's history, age, condition,sex, as well as the severity and type of the medical condition in thesubject, and administration of other pharmaceutically active agents.

As used herein, the term “administer” refers to the placement of acomposition into a subject by a method or route which results in atleast partial localization of the composition at a desired site suchthat desired effect is produced. Routes of administration suitable forthe methods of the invention include both local and systemicadministration. Generally, local administration results in more of theadministered proliferation enhancer (or proliferation enhancer treatedsatellite cells) being delivered to a specific location as compared tothe entire body of the subject, whereas, systemic administration resultsin delivery of the proliferation enhancer (or proliferation enhancertreated satellite cells) to essentially the entire body of the subject.One method of local administration is by intramuscular injection.

In the context of administering a compound treated cell, the term“administering” also include transplantation of such a cell in asubject. As used herein, the term “transplantation” refers to theprocess of implanting or transferring at least one cell to a subject.The term “transplantation” includes, e.g., autotransplantation (removaland transfer of cell(s) from one location on a patient to the same oranother location on the same patient), allotransplantation(transplantation between members of the same species), andxenotransplantation (transplantations between members of differentspecies). Skilled artisan is well aware of methods for implanting ortransplantation of cells for muscle repair and regeneration, which areamenable to the present invention. See for example, U.S. Pat. No.7,592,174 and U.S. Pat. Pub. No. 2005/0249731, content of both of whichis herein incorporated by reference.

Furthermore, the proliferation enhancers can be formulated in the formof ointments, creams powders, or other formulations suitable for topicalformulations. Without wishing to be bound by a theory, theseformulations can deliver the proliferation enhancer from skin to deepermuscle tissue. Accordingly, such formulations can comprise one or moreagents that enhance penetration of active ingredient through skin. Fortopical applications, the proliferation enhancer can be included inwound dressings and/or skin coating compositions.

A proliferation enhancer or composition comprising same can beadministered by any appropriate route known in the art including, butnot limited to, oral or parenteral routes, including intravenous,intramuscular, subcutaneous, transdermal, airway (aerosol), pulmonary,nasal, rectal, and topical (including buccal and sublingual)administration.

Exemplary modes of administration include, but are not limited to,injection, infusion, instillation, inhalation, or ingestion. “Injection”includes, without limitation, intravenous, intramuscular, intraarterial,intrathecal, intraventricular, intracapsular, intraorbital,intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous,subcuticular, intraarticular, sub capsular, subarachnoid, intraspinal,intracerebro spinal, and intrasternal injection and infusion. Inpreferred embodiments of the aspects described herein, the compositionsare administered by intravenous infusion or injection.

As used herein, a “subject” means a human or animal. Usually the animalis a vertebrate such as a primate, rodent, domestic animal or gameanimal. Primates include chimpanzees, cynomologous monkeys, spidermonkeys, and macaques, e.g., Rhesus. Rodents include mice, rats,woodchucks, ferrets, rabbits and hamsters. Domestic and game animalsinclude cows, horses, pigs, deer, bison, buffalo, feline species, e.g.,domestic cat, canine species, e.g., dog, fox, wolf, avian species, e.g.,chicken, emu, ostrich, and fish, e.g., trout, catfish and salmon.Patient or subject includes any subset of the foregoing, e.g., all ofthe above, but excluding one or more groups or species such as humans,primates or rodents. In certain embodiments of the aspects describedherein, the subject is a mammal, e.g., a primate, e.g., a human. Theterms, “patient” and “subject” are used interchangeably herein. Theterms, “patient” and “subject” are used interchangeably herein. Asubject can be male or female.

In certain aspects, the subject does not have or is not otherwiseaffected by acute myeloid leukemia (AML).

Preferably, the subject is a mammal. The mammal can be a human,non-human primate, mouse, rat, dog, cat, horse, or cow, but are notlimited to these examples. Mammals other than humans can beadvantageously used as subjects that represent animal models ofdisorders associated with autoimmune disease or inflammation. Inaddition, the methods and compositions described herein can be used totreat domesticated animals and/or pets.

A subject can be one who has been previously diagnosed with oridentified as suffering from or having a disorder characterized withmuscle damage or muscle atrophy/wasting.

A subject can be one who is not currently being treated with a compounddescribed herein.

A subject can be one who has been previously diagnosed with a diseasethat is being treated with a therapeutic regime comprising compounddescribed herein, wherein the disease is not a disease characterizedwith muscle damage or muscle atrophy/wasting

Accordingly, in some embodiments, the treatment method comprisingadjusting the therapeutic regime of the subject such that at least onesymptom of muscle damage is reduced. Without limitation, a therapeuticregime can be adjusted by modulating the frequency of administration ofthe proliferation enhancer and/or by altering the site or mode ofadministration.

In some embodiments of the aspects described herein, the method furthercomprising diagnosing a subject for muscle damage or muscleatrophy/wasting before treating the subject for muscle repair orregeneration.

In some embodiments of the aspects described herein, the method furthercomprising selecting a subject with muscle damage or muscleatrophy/wasting before treating the subject for muscle repair orregeneration.

A compound described herein can be co-administrated to a subject incombination with a pharmaceutically active agent. Exemplarypharmaceutically active compound include, but are not limited to, thosefound in Harrison's Principles of Internal Medicine, 13^(th) Edition,Eds. T. R. Harrison et al. McGraw-Hill N.Y., N.Y.; Physicians' DeskReference, 50^(th) Edition, 1997, Oradell N.J., Medical Economics Co.;Pharmacological Basis of Therapeutics, 8^(th) Edition, Goodman andGilman, 1990; United States Pharmacopeia, The National Formulary, USPXII NF XVII, 1990; current edition of Goodman and Oilman's ThePharmacological Basis of Therapeutics; and current edition of The MerckIndex, the complete content of all of which are herein incorporated inits entirety.

In some embodiments of the aspects described herein, thepharmaceutically active agent is a growth factor. Exemplary growthfactors include, but are not limited to, basic epidermal growth factor(bEGF), fibroblast growth factors (FGF), FGF-1, FGF-2 (bFGF), FGF-4,thymosins, platelet-derived growth factors (PDGF), insulin bindinggrowth factors (IGF), IGF-1, IGF-2, epidermal growth factor (EGF),transforming growth factor (TGF), TGF-alpha, TGF-beta, cartilageinducing factors-A and —B, osteoid-inducing factors, osteogenin, bonemorphogenic proteins, and other bone growth factors, collagen growthfactors, heparin-binding growth factor-1 or -2, and their biologicallyactive derivatives.

The proliferation enhancer and the pharmaceutically active agent can beadministrated to the subject in the same pharmaceutical composition orin different pharmaceutical compositions (at the same time or atdifferent times). When administrated at different times, theproliferation enhancer and the pharmaceutically active agent can beadministered within 5 minutes, 10 minutes, 20 minutes, 60 minutes, 2hours, 3 hours, 4, hours, 8 hours, 12 hours, 24 hours of administrationof the other. When the proliferation enhancer and the pharmaceuticallyactive agent are administered in different pharmaceutical compositions,routes of administration can be different.

The amount of the proliferation enhancer that can be combined with acarrier material to produce a single dosage form will generally be thatamount of the proliferation enhancer that produces a therapeutic effect.Generally out of one hundred percent, this amount will range from about0.01% to 99% of the compound, preferably from about 5% to about 70%,most preferably from 10% to about 30%.

Toxicity and therapeutic efficacy can be determined by standardpharmaceutical procedures in cell cultures or experimental animals,e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compositions that exhibit large therapeutic indices, are preferred.

As used herein, the term ED denotes effective dose and is used inconnection with animal models. The term EC denotes effectiveconcentration and is used in connection with in vitro models.

The data obtained from the cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofsuch compounds lies preferably within a range of circulatingconcentrations that include the ED50 with little or no toxicity. Thedosage may vary within this range depending upon the dosage formemployed and the route of administration utilized.

The therapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range that includes the IC50 (i.e., theconcentration of the therapeutic which achieves a half-maximalinhibition of symptoms) as determined in cell culture. Levels in plasmamay be measured, for example, by high performance liquid chromatography.The effects of any particular dosage can be monitored by a suitablebioassay.

The dosage may be determined by a physician and adjusted, as necessary,to suit observed effects of the treatment. Generally, the compositionsare administered so that the proliferation enhancer is given at a dosefrom 1 μg/kg to 150 mg/kg, 1 μg/kg to 100 mg/kg, 1 μg/kg to 50 mg/kg, 1μg/kg to 20 mg/kg, 1 μg/kg to 10 mg/kg, 1 μg/kg to 1 mg/kg, 100 μg/kg to100 mg/kg, 100 μg/kg to 50 mg/kg, 100 μg/kg to 20 mg/kg, 100 μg/kg to 10mg/kg, 100 μg/kg to 1 mg/kg, 1 mg/kg to 100 mg/kg, 1 mg/kg to 50 mg/kg,1 mg/kg to 20 mg/kg, 1 mg/kg to 10 mg/kg, 10 mg/kg to 100 mg/kg, 10mg/kg to 50 mg/kg, or 10 mg/kg to 20 mg/kg. It is to be understood thatranges given here include all intermediate ranges, for example, therange 1 tmg/kg to 10 mg/kg includes 1 mg/kg to 2 mg/kg, 1 mg/kg to 3mg/kg, 1 mg/kg to 4 mg/kg, 1 mg/kg to 5 mg/kg, 1 mg/kg to 6 mg/kg, 1mg/kg to 7 mg/kg, 1 mg/kg to 8 mg/kg, 1 mg/kg to 9 mg/kg, 2 mg/kg to 10mg/kg, 3 mg/kg to 10 mg/kg, 4 mg/kg to 10 mg/kg, 5 mg/kg to 10 mg/kg, 6mg/kg to 10 mg/kg, 7 mg/kg to 10 mg/kg, 8 mg/kg to 10 mg/kg, 9 mg/kg to10 mg/kg, and the like. It is to be further understood that the rangesintermediate to the given above are also within the scope of thisinvention, for example, in the range 1 mg/kg to 10 mg/kg, dose rangessuch as 2 mg/kg to 8 mg/kg, 3 mg/kg to 7 mg/kg, 4 mg/kg to 6 mg/kg, andthe like.

In some embodiments, the compositions are administered at a dosage sothat the proliferation enhancer or a metabolite thereof has an in vivoconcentration of less than 500 nM, less than 400 nM, less than 300 nM,less than 250 nM, less than 200 nM, less than 150 nM, less than 100 nM,less than 50 nM, less than 25 nM, less than 20, nM, less than 10 nM,less than 5 nM, less than 1 nM, less than 0.5 nM, less than 0.1 nM, lessthan 0.05, less than 0.01, nM, less than 0.005 nM, less than 0.001 nMafter 15 mins, 30 mins, 1 hr, 1.5 hrs, 2 hrs, 2.5 hrs, 3 hrs, 4 hrs, 5hrs, 6 hrs, 7 hrs, 8 hrs, 9 hrs, 10 hrs, 11 hrs, 12 hrs or more of timeof administration.

With respect to duration and frequency of treatment, it is typical forskilled clinicians to monitor subjects in order to determine when thetreatment is providing therapeutic benefit, and to determine whether toincrease or decrease dosage, increase or decrease administrationfrequency, discontinue treatment, resume treatment or make otheralteration to treatment regimen. The dosing schedule can vary from oncea week to daily depending on a number of clinical factors, such as thesubject's sensitivity to the polypeptides. The desired dose can beadministered everyday or every third, fourth, fifth, or sixth day. Thedesired dose can be administered at one time or divided into subdoses,e.g., 2-4 subdoses and administered over a period of time, e.g., atappropriate intervals through the day or other appropriate schedule.Such sub-doses can be administered as unit dosage forms. In someembodiments of the aspects described herein, administration is chronic,e.g., one or more doses daily over a period of weeks or months. Examplesof dosing schedules are administration daily, twice daily, three timesdaily or four or more times daily over a period of 1 week, 2 weeks, 3weeks, 4 weeks, 1 month, 2 months, 3 months, 4 months, 5 months, or 6months or more.

Screening Assays

In yet another aspect, the invention provides for a method of screeningfor a candidate compound for stimulating or increasing proliferation ina satellite cell population, the method comprising:

-   -   (a) contacting a population of satellite cells with a test        compound;    -   (b) assessing satellite proliferation; and    -   (c) selecting the compound that induces, increases or enhances        satellite cell proliferation.

As used herein, the term “test compound” refers to compounds and/orcompositions that are to be screened for their ability to induce,stimulate, enhance, or increase satellite cell proliferation. The testcompounds can include a wide variety of different compounds, includingchemical compounds and mixtures of chemical compounds, e.g., smallorganic or inorganic molecules; saccharines; oligosaccharides;polysaccharides; biological macromolecules, e.g., peptides, proteins,and peptide analogs and derivatives; antibodies, antibodies fragments,peptidomimetics; nucleic acids; nucleic acid analogs and derivatives; anextract made from biological materials such as bacteria, plants, fungi,or animal cells; animal tissues; naturally occurring or syntheticcompositions; and any combinations thereof.

In some embodiments, the test compound is a small molecule.

The number of possible test compounds runs into millions. Methods fordeveloping small molecule, polymeric and genome based libraries aredescribed, for example, in Ding, et al. J Am. Chem. Soc. 124: 1594-1596(2002) and Lynn, et al., J. Am. Chem. Soc. 123: 8155-8156 (2001).Commercially available compound libraries can be obtained from, e.g.,ArQule, Pharmacopia, graffinity, Panvera, Vitas-M Lab, BiomolInternational and Oxford. These libraries can be screened using thescreening devices and methods described herein. Chemical compoundlibraries such as those from NIH Roadmap, Molecular Libraries ScreeningCenters Network (MLSCN) can also be used. A comprehensive list ofcompound libraries can be found atbroad.harvard.edu/chembio/platform/screening/compound_libraries/index. Achemical library or compound library is a collection of stored chemicalsusually used ultimately in high-throughput screening or industrialmanufacture. The chemical library can consist in simple terms of aseries of stored chemicals. Each chemical has associated informationstored in some kind of database with information such as the chemicalstructure, purity, quantity, and physiochemical characteristics of thecompound.

Depending upon the particular embodiment being practiced, the testcompounds can be provided free in solution, or may be attached to acarrier, or a solid support, e.g., beads. A number of suitable solidsupports may be employed for immobilization of the test compounds.Examples of suitable solid supports include agarose, cellulose, dextran(commercially available as, i.e., Sephadex, Sepharose) carboxymethylcellulose, polystyrene, polyethylene glycol (PEG), filter paper,nitrocellulose, ion exchange resins, plastic films,polyaminemethylvinylether maleic acid copolymer, glass beads, amino acidcopolymer, ethylene-maleic acid copolymer, nylon, silk, etc.Additionally, for the methods described herein, test compounds may bescreened individually, or in groups. Group screening is particularlyuseful where hit rates for effective test compounds are expected to below such that one would not expect more than one positive result for agiven group.

In some embodiments, the test compound induces, enhances, or increasessatellite cell proliferation by at least 5%, 10%, 20%, 30%, 40%, 50%,50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold, 2-fold, 3-fold, 4-fold,5-fold, 10-fold, 50-fold, 100-fold or more relative to an untreatedcontrol.

In some embodiments, the step of assessing satellite cell proliferationcomprises detecting a satellite cell marker.

In some embodiments, the step of assessing satellite cell proliferationcomprises detecting a satellite cell marker and a cell replicationmarker. A selected test compound can be further limited to the compoundwhere the satellite cell marker and the cell-replication markerco-localize in the same cell.

Increased or enhanced satellite proliferation can be assessed by: (i)increased total number of cells in the culture, as compared to anuntreated control; (ii) increased total number of cells expressing atleast one satellite cell marker in the culture, as compared to anuntreated control; (iii) increased ratio of cells expressing at leastone satellite cell marker to the total number of cells in the culture,as compared to an untreated control; (iv) increased number of cellsexpressing at least one cell-replication marker, as compared to anuntreated control; (v) increased ratio of cells expressing at least onecell-replication marker, as compared to an untreated control; or (vi) acombination thereof.

In some embodiments, satellite cell proliferation is assessed viaautomated image acquisition and analysis using a Cellomics ArrayScanVTI. The acquisition thresholds/parameters are established such that thecomputer-based calls of replication events are consistent withhuman-based calls. Such automated image acquisition and analysis allowsfor high-throughput screening of compounds.

Generally plating density can range from about 10 k cells/well to about100 k cells/well. In some embodiments, cellular plating density is inthe range from about 25 k cells/well to about 75 k cells/well. In oneembodiment, cellular plating density is about 60 k cells/well.Generally, at least 75%, 80%, 85%, 90%, 95% or more of the cells areviable at time of plating.

After plating, satellite cells can be allowed to adhere to the surfacefor a sufficient time, e.g. at least at least 1 hour, 2 hours, 3, hours,4 hours, 6 hours, 8 hours, 12 hours, 24 hours, 36 hours, 48 hours ormore, before contacting with the test compound. In some embodiments, thecells are allowed to adhere for 48 hours before compound treatment.After the cells have been allowed to adhere for a sufficient time, themedia can be changed before treatment with compound of interest.

Generally, compounds can be tested at any concentration that can enhancereplication of β-cells relative to a control over an appropriate timeperiod. In some embodiments, compounds are tested at concentration inthe range of about 0.1 nM to about 1000 mM. Preferably the compound istested in the range of about 0.1 μM to about 20 μM, about 0.1 μM toabout 10 μM, or about 0.1 μM to about 5 μM. In one embodiment, compoundsare tested at 1 μM.

The satellite cell population can be maintained at any temperaturesuitable for satellite cell cultures. In one embodiment, the satellitecells are maintained at a temperature in the range of about 15° C. toabout 55° C. In one embodiment, the pancreatic cells are maintained at37° C.

Generally, the number of satellite cells in the culture can be countedafter the satellite cells have been in contact with the test compoundfor a sufficient time, e.g., at least 1 hour, at least 2 hours, at least3, hours, at least 4 hours, at least 5 hours, at least 6 hours, at least7 hours, at least 8 hours, at least 9 hours, at least 10 hours, at least11 hours, at least 12 hours, at least 24 hours, at least 36 hours, atleast 48 hours, at least 5 days, at least 1 week, at least 2 weeks, atleast 3 weeks, or more. The cells can be counted manually or by anautomated system. Use of an automated system allows for high-throughputscreening of compounds.

The inventors have discovered that in some instances prolonged treatmentwith the proliferation enhancer does not lead to higher satellite cellproliferation as compared to treatment for a shorter period of time.Accordingly, the number of satellite cells in the culture can be countedafter the satellite cells have been in contact with the test compoundfor between 1 hour and seven days. For example, the number of satellitecells in the culture can be counted after the satellite cells have beenin contact with the test compound for one, two, three, four, five or sixdays.

Satellite-cell and replication cell marker detection can be done afterthe satellite cells are in contact with the test compound for asufficient time, e.g., at least 1 hour, at least 2 hours, at least 3,hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 7hours, at least 8 hours, at least 9 hours, at least 10 hours, at least11 hours, at least 12 hours, at least 24 hours, at least 36 hours, atleast 48 hours, at least 5 days, at least 1 week, at least 2 weeks, atleast 3 weeks, or more. After marker detection, number of cellsexpressing cell-replication and/or satellite cell marker can be counted.Marker detection can include the steps of preparing the cells for theappropriate assay, e.g., fixing and/or staining the cells.

In some embodiments, the satellite cell and cell replication markerdetection can be done after the satellite cells are in contact with thetest compound for 1 hour to about 7 days.

In some embodiments, the satellite cell and cell replication markerdetection can be done after the satellite cells are in contact with thetest compound for one, two, three, four, five or six days.

In some embodiments, the method comprises additionally selecting thecompound that increased the ratio of satellite cells to the total numberof cells as compared to an untreated control.

The term “satellite-cell marker” refers to, without limitation,proteins, peptides, nucleic acids, polymorphism of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analytes which are specifically expressed or presentin satellite cells. Exemplary satellite cell markers include, but arenot limited to, PAX7, PAX3, Myf5, MyoD, and desmin. Other markers thatcan be used include, but are not limited to, beta-integrin 1 and CXCR4.Method of identifying satellite cells is also described in Sherwood etal. (Cell, 2004, 119: 543-554), content of which is incorporated hereinby reference.

The terms “cell replication marker” refers to, without limitation,proteins, peptides, nucleic acids, polymorphisma of proteins and nucleicacids, splice variants, fragments of proteins or nucleic acids,elements, and other analytes which are specifically associated with cellproliferation. Additionally, “cell-replication marker”, includesenzymatic activity when changes, e.g., increase or decrease, in theenzymatic activity are specifically associated with cell proliferation.Exemplary cell replication markers include, but are not limited to,phosphorylated histone H3 (PH3), Ki-67 protein, phosphorylated MPM-2antigen, Proliferating Cell Nuclear Antigen (PCNA, a protein that isexpressed in the nuclei of cells during the DNA synthesis phase of thecell cycle), phospho-S780-Rb epitope (Jacobberger, J W, et al. CytometryA (2007), 73A:5-15), Cenp-F (mitosin), class III β-Tublin, spindalcheckpint protine hMad2, phosphorylated myosin light chain kinase,topoisomerase II, Check point kinase 1 (Chk1), Vesicular MonoamineTransporter 2 (VMAT2), loss of cyclin-dependent kinase 1 (Cdk1) kinaseactivity. Histone H3 can be phosphorylated at Ser28 or Ser10.

Cell replication markers and satellite cell markers can be detected bymethods known in the art and easily available to the skilled artisan,for example appropriate ELISA, immunofluorescent, or immunohistochemicalassays can be used for detection. MIB-1 is a commonly used monoclonalantibody that detects the Ki-67 protein. It is used in clinicalapplications to determine the Ki-67 labelling index. Ki-67 ELISA aredescribed in Klein, C L, et al., J. Mater. Sci. Mater. Med. (2000),11:125-132; Frahm, S O, et al., J. Immunol. Methods (199*0, 211:43-50;and Key G, et al., J. Immunol. Methods (1994), 177:113-117.Phospho-Histone H3 antibodies for detection of phosphorylated Histone H3are commercially available from Cell Signaling Technology and Millipore.Antibodies against PCNA are commercially available from Sigma Aldrich.Antibodies to MPM-2 antigen are specific for cells in mitosis,recognizes a family of proteins that share a common phosphorylatedepitope.

In some embodiments of this and other aspects described herein, thesatellite cell can be isolated from a mammal. Methods for isolatingcells from a subject are described, for example in Sherwood et al.(Cell, 2004, 119: 543-554), content of which is incorporated herein byreference.

In some embodiments of this and other aspects described herein, thesatellite cell can be isolated from a mouse.

A satellite cell can be transformed cell. As used herein, the term“transformed cells” is art recognized and refers to cells which haveconverted to a state of unrestrained growth, i.e., they have acquiredthe ability to grow through an indefinite number of divisions inculture. Transformed cells may be characterized by such terms asneoplastic, anaplastic and/or hyperplastic, with respect to their lossof growth control. In general, term “transformed satellite cell” refersto satellite cells which exhibit increased capacity to persist in serialsubcultures or increased growth rate in vitro.

In some embodiments, the screening method is a high-throughputscreening. High-throughput screening (HTS) is a method for scientificexperimentation that uses robotics, data processing and controlsoftware, liquid handling devices, and sensitive detectors.High-Throughput Screening or HTS allows a researcher to quickly conductmillions of biochemical, genetic or pharmacological tests.High-Throughput Screening are well known to one skilled in the art, forexample, those described in U.S. Pat. Nos. 5,976,813; 6,472,144;6,692,856; 6,824,982; and 7,091,048, and contents of each of which isherein incorporated by reference in its entirety.

HTS uses automation to run a screen of an assay against a library ofcandidate compounds. An assay is a test for specific activity: usuallyinhibition or stimulation of a biochemical or biological mechanism.Typical HTS screening libraries or “decks” can contain from 100,000 tomore than 2,000,000 compounds.

The key labware or testing vessel of HTS is the microtiter plate: asmall container, usually disposable and made of plastic, that features agrid of small, open divots called wells. Modern microplates for HTSgenerally have either 384, 1536, or 3456 wells. These are all multiplesof 96, reflecting the original 96 well microplate with 8×12 9 mm spacedwells.

To prepare for an assay, the researcher fills each well of the platewith the appropriate reagents that he or she wishes to conduct theexperiment with, such as a satellite cell population. After someincubation time has passed to allow the reagent to absorb, bind to, orotherwise react (or fail to react) with the compounds in the wells,measurements are taken across all the plate's wells, either manually orby a machine. Manual measurements are often necessary when theresearcher is using microscopy to (for example) seek changes that acomputer could not easily determine by itself. Otherwise, a specializedautomated analysis machine can run a number of experiments on the wellssuch as colorimetric measurements, radioactivity counting, etc. In thiscase, the machine outputs the result of each experiment as a grid ofnumeric values, with each number mapping to the value obtained from asingle well. A high-capacity analysis machine can measure dozens ofplates in the space of a few minutes like this, generating thousands ofexperimental data points very quickly.

By way of example, in one embodiment, the assay was performed as follow.Satellite cells were harvested from CAG-B-actin-GFP mice, aged 2-4months old via the FACS isolation method outlined in Sherwood et al 2004and Cerletti et al 2008. Briefly, all limb muscles and abdominal muscleswere harvested from the animals and digested first in a 0.2% collagenasesolution, then in a 0.0125% collagenase/0.05% dispase solution. Thefiltered solution was stained for cell surface markers, and put throughFACS. Cells that were negative for Mac1, Sca1 and CD45 and positive forBeta-integrin 1 and CXCR4 were used for the screening assay. Cells wereplated directly from the FACS machine at 50 cells/well, into 96-wellplates coated with 10 ug/mL laminin (4-6 hours at 37 C, then partiallyremoved). Media for the screen was Ham's F-10 supplemented with 10%heat-inactivated horse serum, 1× penicillin/streptomycin and 1×L-glutamine. Basic FGF (bFGF) was added only to the positive controlwells; all other wells received media only. The day of plating wascalled Day 0. The day after plating, Day 1, compounds were added. Allcompounds were dissolved in DMSO, and initially tested at 10 uM, 1 uM or0.1 uM in duplicate. The negative control for each plate was the sameconcentration of DMSO (no compounds dissolved). Compounds were incubatedwith the cells until Day 4, with no media change. bFGF was spiked intopositive control wells daily. On Day 4, plates were fixed in 4%paraformaldehyde and washed with phosphate buffered saline. Plates wereimaged directly on the Opera Confocal imager, since the cells werereadily visible from the CAG-EGFP-Beta-actin transgene. An Acapellascript was used to count the number of cells in each well. Wells werescored for proliferation based on the cell counts (DMSO-treated wellsusually had around 50 cells at the end of the assay, bFGF-treated wellsusually had 150-250 cells at the end). Inventors selected compounds ashits if they had cell numbers greater than or equal to one (or, for thesecond session of screening, three) standard deviation(s) from the DMSOcontrols. Compounds thus selected were then tested in an initial dosecurve, usually concentrations between 20 nM-30 uM commensurate with theconcentration flagged as a hit. A compound was active if it was able tocause proliferation to cell numbers at least two standard deviationsover the DMSO negative control. Any compounds that were found to beactive in the dose curve were re-supplied from the original vendors.Re-supplied compounds were again tested in a dose curve forproliferation ability. Compounds still found to be active were then putin the queue for optimization and characterization.

In another aspect, the invention provides a compound selected by thescreening assay described herein. It is to be understood that analogs,derivatives, and isomers of the compounds selected by the screeningassays described herein are also claimed herein.

Kits

In another aspect, the invention provides a kit for muscle repair orregeneration. In some embodiments, the kit comprises a compounddescribed herein, e.g., a compound selected from the group consisting ofkinase inhibitors, G protein coupled receptor (GPCR) modulators, histonedeacetylases (HDAC) modulators, hedgehog signaling pathway modulators,neuropeptides, dopamine receptor modulators, serotonin receptormodulators, histamine receptor modulators, ionophores, ion channelmodulators, gamma-secretase modulators, and any combinations thereof.The compound can be pre-formulated into a pharmaceutical formulation foradministration or ingredients for formulating into a pharmaceuticalformulation can be provided in the kit.

In some embodiments, the kit comprises a compound described herein,wherein the compound is formulated for topical application.

In some embodiments, the kit comprises a population of satellite cells,wherein at least one cell in the population has been pretreated bycontacting the cells with a compound described herein.

In addition to the above mentioned components, the kit can includeinformational material. The informational material can be descriptive,instructional, marketing or other material that relates to the methodsdescribed herein and/or the use of the compound for the methodsdescribed herein. For example, the informational material describesmethods for administering the formulation to a subject. The kit can alsoinclude a delivery device.

In one embodiment, the informational material can include instructionsto administer the formulation in a suitable manner, e.g., in a suitabledose, dosage form, or mode of administration (e.g., a dose, dosage form,or mode of administration described herein). In another embodiment, theinformational material can include instructions for identifying asuitable subject, e.g., a human, e.g., an adult human. The informationalmaterial of the kits is not limited in its form. In many cases, theinformational material, e.g., instructions, is provided in printedmatter, e.g., a printed text, drawing, and/or photograph, e.g., a labelor printed sheet. However, the informational material can also beprovided in other formats, such as Braille, computer readable material,video recording, or audio recording. In another embodiment, theinformational material of the kit is a link or contact information,e.g., a physical address, email address, hyperlink, website, ortelephone number, where a user of the kit can obtain substantiveinformation about the formulation and/or its use in the methodsdescribed herein. Of course, the informational material can also beprovided in any combination of formats.

In some embodiments the individual components of the formulation can beprovided in one container. Alternatively, it can be desirable to providethe components of the formulation separately in two or more containers,e.g., one container for an oligonucleotide preparation, and at leastanother for a carrier compound. The different components can becombined, e.g., according to instructions provided with the kit. Thecomponents can be combined according to a method described herein, e.g.,to prepare and administer a pharmaceutical composition.

In addition to the formulation, the composition of the kit can includeother ingredients, such as a solvent or buffer, a stabilizer or apreservative, and/or a second agent for treating a condition or disorderdescribed herein. Alternatively, the other ingredients can be includedin the kit, but in different compositions or containers than theformulation. In such embodiments, the kit can include instructions foradmixing the formulation and the other ingredients, or for using theoligonucleotide together with the other ingredients.

The compound can be provided in any form, e.g., liquid, dried orlyophilized form. It is preferred that the formulation be substantiallypure and/or sterile. When the formulation is provided in a liquidsolution, the liquid solution preferably is an aqueous solution, with asterile aqueous solution being preferred. When the formulation isprovided as a dried form, reconstitution generally is by the addition ofa suitable solvent. The solvent, e.g., sterile water or buffer, canoptionally be provided in the kit.

In some embodiments, the kit contains separate containers, dividers orcompartments for the formulation and informational material. Forexample, the formulation can be contained in a bottle, vial, or syringe,and the informational material can be contained in a plastic sleeve orpacket. In other embodiments, the separate elements of the kit arecontained within a single, undivided container. For example, theformulation is contained in a bottle, vial or syringe that has attachedthereto the informational material in the form of a label.

In some embodiments, the kit includes a plurality, e.g., a pack, ofindividual containers, each containing one or more unit dosage forms ofthe formulation. For example, the kit includes a plurality of syringes,ampules, foil packets, or blister packs, each containing a single unitdose of the formulation. The containers of the kits can be air tightand/or waterproof.

Exemplary embodiments of can also be described by one or more of thefollowing numbered paragraphs.

-   1. A method of increasing satellite cell proliferation, the method    comprising: contacting a satellite cell with a compound selected    from the group consisting of kinase inhibitors, G protein coupled    receptor (GPCR) modulators, histone deacetylases (HDAC) modulators,    hedgehog signaling pathway modulators, neuropeptides, dopamine    receptor modulators, serotonin receptor modulators, histamine    receptor modulators, adenosine receptor agonists, ionophores, ion    channel modulators, gamma-secretase modulators, corticosteroids, and    any combination thereof.-   2. The method of paragraph 1, wherein the compound is selected from    the group consisting of small organic or inorganic molecules;    saccharines; oligosaccharides; polysaccharides; peptides, proteins,    peptide analogs and derivatives; antibodies, antibodies fragments,    peptidomimetics; nucleic acids; nucleic acid analogs and    derivatives; an extract made from biological materials; naturally    occurring or synthetic compositions; and any combination thereof.-   3. The method of any of paragraphs 1-2, wherein the compound is a    Flt3 kinase, PDGFR/EGFR, Bcr-abl, Jak3, or SRC kinase inhibitor.-   4. The method of any of paragraphs 1-3, wherein the compound is    selected from the group consisting of a protein kinase inhibitor and    a receptor kinase inhibitor.-   5. In some embodiments, the kinase inhibitor can be selected from    the group

and any combinations thereof.

-   6. The method of any of paragraphs 1-4, wherein the compound is    contacted with the satellite cell at a concentration of about 0.01    nM to about 100 μM.-   7. The method of any of paragraphs 1-5, wherein said contacting is    for at least 1 hour.-   8. The method of any of paragraphs 1-6, wherein said contacting is    for one to seven days.-   9. The method of any of paragraphs 1-7, wherein the contact is in    vitro.-   10. The method of any of paragraphs 1-8, wherein the contact is ex    vivo.-   11. The method of any of paragraphs 1-9, wherein the contact is in    vivo.-   12. The method of paragraph 10, wherein in vivo contact is in a    mammal.-   13. The method of paragraph 10 or 11, wherein in vivo contact is in    a human.-   14. The method of any of paragraphs 10-12, wherein the in vivo    contact is in a subject, where the subject is in need of treatment    for damaged muscle tissue.-   15. The method of paragraph 13, wherein the damaged muscle tissue is    the result of a physical injury or accident, disease, infection,    over-use, loss of blood circulation, or muscle atrophy or wasting.-   16. The method of paragraph 13 or 14, wherein the damaged muscle    tissue is dystrophic muscle or an ageing muscle.-   17. The method of any of paragraphs 13-15, wherein the damaged    muscle tissue is the result of muscle atrophy/wasting.-   18. A method for muscle repair or regeneration in a subject, the    method comprising administering a therapeutically effective amount    of a compound to the subject, which subject has a damaged muscle    tissue, and wherein the compound is selected from the group    consisting of kinase inhibitors, G protein coupled receptor (GPCR)    modulators, histone deacetylases (HDAC) modulators, hedgehog    signaling pathway modulators, neuropeptides, dopamine receptor    modulators, serotonin receptor modulators, histamine receptor    modulators, ionophores, ion channel modulators, gamma-secretase    modulators, and any combination thereof.-   19. The method of paragraph 17, wherein the damaged muscle tissue is    the result of a physical injury or accident, disease, infection,    over-use, loss of blood circulation, or muscle atrophy or wasting.-   20. The method of any of paragraphs 17-18, wherein the damaged    muscle tissue is dystrophic muscle or an ageing muscle.-   21. The method of any of paragraphs 17-19, wherein the damaged    muscle tissue is the result of muscle atrophy/wasting.-   22. The method of any of paragraphs 17-20, wherein the subject is a    mammal.-   23. The method of any of paragraphs 17-21, wherein the subject is    human.-   24. The method of any of paragraphs 17-22, wherein the compound is    co-administered with a therapeutic agent.-   25. The method of paragraph 23, wherein the compound and the    therapeutic agent are administered in the same formulation.-   26. The method of any of paragraphs 17-24, wherein the compound is    administered at a dosage of from 1 μg/kg to 150 mg/kg.-   27. The method of any of paragraphs 17-25, wherein said    administering is by injection, infusion, instillation, inhalation,    or ingestion.-   28. The method of any of paragraphs 17-26, wherein said    administering is once daily.-   29. The method of any of paragraphs 17-27, further comprising    diagnosing the subject for muscle damage or muscle atrophy/wasting    before treating the subject for muscle repair or regeneration.-   30. The method of any of paragraphs 17-28, wherein the compound is    selected from the group consisting of

and any combinations thereof.

-   31. A high throughput assay for screening compounds that induce,    stimulate, enhance or increase satellite proliferation, the assay    comprising:    -   (a) contacting a satellite cell with a test compound;    -   (b) assessing satellite cell proliferation; and    -   (c) selecting the compound that induces, stimulates, enhances or        increases satellite cell replication or growth-   32. The assay of paragraph 30, wherein the step of assessing    satellite cell proliferation comprises detecting a cell marker.-   33. The assay of paragraph 31, wherein the cell marker is selected    from the group consisting of CXCR4, β1-integrin, Sca-1, Mac-1, CD45,    PAX7, PAX3, Myf5, MyoD, desmin, and any combinations thereof.-   34. The assay of any of paragraphs 30-32, wherein the test compound    has a concentration in the range of 0.1 nM to 1000 mM.-   35. The assay of any of paragraphs 30-33, wherein the assay is    performed at a temperature in the range of about 15° C. to about 55°    C.-   36. The assay of any of paragraphs 30-34, wherein the test compound    is contacted with the pancreatic cells for 1 hour to seven days-   37. The assay of any of paragraphs 30-35, wherein the test compound    increases satellite cell proliferation by at least 5%, 10%, 20%,    30%, 40%, 50%, 50%, 70%, 80%, 90%, 1-fold, 1.1-fold, 1.5-fold,    2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 50-fold, 100-fold or more    higher relative to an untreated control.-   38. The assay of any of paragraphs 30-36, wherein the satellite    cells are isolated from a mammal.-   39. The assay of any of paragraphs 30-37, wherein the satellite    cells are isolated from a subject, where the subject is in need of    treatment for damaged muscle tissue.-   40. The method of paragraph 38, wherein the damaged muscle tissue is    the result of a physical injury or accident, disease, infection,    over-use, loss of blood circulation, or muscle atrophy or wasting.-   41. The method of paragraph 38 or 49, wherein the damaged muscle    tissue is dystrophic muscle or an ageing muscle.-   42. The method of any of paragraphs 38-40, wherein the damaged    muscle tissue is the result of muscle atrophy/wasting.-   43. In some embodiments of any of the foregoing paragraphs 1-42, the    kinase inhibitor comprises

or a salt, ester or chelate thereof.

-   44. A method of increasing satellite cell proliferation, the method    comprising: contacting a satellite cell with a compound, wherein the    compound is a kinase inhibitor.-   45. The method of paragraph 44, wherein the kinase is a protein    kinase.-   46. The method of paragraph 44, wherein the protein kinase is a    protein tyrosine kinase.-   47. The method of paragraph 46, wherein the protein tyrosine kinase    is a receptor protein tyrosine kinase.-   48. The method of paragraph 47, wherein the receptor protein    tyrosine kinase is a member of the RET family.-   49. The method of paragraph 47, wherein the receptor protein    tyrosine kinase is RET.-   50. The method of paragraph 47, wherein the compound interferes with    binding of a ligand to the receptor protein tyrosine kinase.-   51. The method of paragraph 44, wherein the compound is selected    from the group consisting of small organic or inorganic molecules,    saccharines, oligosaccharides, polysaccharides, peptides, proteins,    peptide analogs and derivatives, antibodies, antibodies fragments,    peptidomimetics, nucleic acids, nucleic acid analogs and    derivatives, an extract made from biological materials, naturally    occurring or synthetic compositions and any combination thereof.-   52. The method of paragraph 51, wherein the compound is an antibody.-   53. The method of paragraph 44, wherein the compound is contacted    with the satellite cell at a concentration of about 0.01 nM to about    100 μM.-   54. The method of paragraph 44, wherein the contact is in vivo.-   55. The method of paragraph 54, wherein the in vivo contact is in a    mammal.-   56. The method of paragraph 55, wherein the in vivo contact is in a    mammal, and wherein the mammal is in need of treatment for damaged    muscle tissue.-   57. The method of paragraph 56, wherein the damaged muscle tissue is    a result of a physical injury or accident, disease, infection,    over-use, loss of blood circulation, muscle atrophy, muscle wasting,    dystrophic muscle or ageing muscle.-   58. The method of any of paragraphs 44-57, wherein the compound    comprises quizartinib (AC220), or any salt, ester or chelate    thereof.-   59. A method for muscle repair or muscle regeneration in a subject    having damaged muscle tissue, the method comprising administering a    therapeutically effective amount of a compound to the subject,    wherein the compound is a kinase inhibitor.-   60. The method of paragraph 59, wherein the kinase is a protein    kinase.-   61. The method of paragraph 60, wherein the protein kinase is a    protein tyrosine kinase.-   62. The method of paragraph 61, wherein the protein tyrosine kinase    is a receptor protein tyrosine kinase.-   63. The method of paragraph 62, wherein the receptor protein    tyrosine kinase is a member of the RET family.-   64. The method of paragraph 61, wherein the receptor protein    tyrosine kinase is RET.-   65. The method of paragraph 61, wherein the compound interferes with    binding of a ligand to the receptor protein tyrosine kinase.-   66. The method of paragraph 59, wherein the compound is selected    from the group consisting of small organic or inorganic molecules,    saccharines, oligosaccharides, polysaccharides, peptides, proteins,    peptide analogs and derivatives, antibodies, antibodies fragments,    peptidomimetics, nucleic acids, nucleic acid analogs and    derivatives, an extract made from biological materials, naturally    occurring or synthetic compositions and any combination thereof.-   67. The method of paragraph 59, wherein the damaged muscle tissue is    the result of a physical injury or accident, disease, infection,    over-use, loss of blood circulation, muscle atrophy, muscle wasting,    dystrophic muscle or ageing muscle.-   68. The method of paragraph 59, wherein the subject is a mammal.-   69. The method of any of paragraphs 59-68, wherein the compound    comprises quizartinib (AC220), or any salt, ester or chelate thereof-   70. The method of any paragraphs 44-69, wherein the compound is    selected from the group consisting of

and any combinations thereof.

Some Selected Definitions

For convenience, certain terms employed herein, in the specification,examples and appended claims are collected herein. Unless statedotherwise, or implicit from context, the following terms and phrasesinclude the meanings provided below. Unless explicitly stated otherwise,or apparent from context, the terms and phrases below do not exclude themeaning that the term or phrase has acquired in the art to which itpertains. The definitions are provided to aid in describing particularembodiments, and are not intended to limit the claimed invention,because the scope of the invention is limited only by the claims.Further, unless otherwise required by context, singular terms shallinclude pluralities and plural terms shall include the singular.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as those commonly understood to one of ordinaryskill in the art to which this invention pertains. Although any knownmethods, devices, and materials may be used in the practice or testingof the invention, the methods, devices, and materials in this regard aredescribed herein.

As used herein the term “comprising” or “comprises” is used in referenceto compositions, methods, and respective component(s) thereof, that areessential to the invention, yet open to the inclusion of unspecifiedelements, whether essential or not.

The singular terms “a,” “an,” and “the” include plural referents unlesscontext clearly indicates otherwise. Similarly, the word “or” isintended to include “and” unless the context clearly indicatesotherwise.

Other than in the operating examples, or where otherwise indicated, allnumbers expressing quantities of ingredients or reaction conditions usedherein should be understood as modified in all instances by the term“about.” The term “about” when used in connection with percentages maymean±5% of the value being referred to. For example, about 100 meansfrom 95 to 105.

Although methods and materials similar or equivalent to those describedherein can be used in the practice or testing of this disclosure,suitable methods and materials are described below. The term “comprises”means “includes.” The abbreviation, “e.g.” is derived from the Latinexempli gratia, and is used herein to indicate a non-limiting example.Thus, the abbreviation “e.g.” is synonymous with the term “for example.”

The terms “decrease”, “reduced”, “reduction”, “decrease” or “inhibit”are all used herein generally to mean a decrease by a statisticallysignificant amount. However, for avoidance of doubt, “reduced”,“reduction” or “decrease” or “inhibit” means a decrease by at least 10%as compared to a reference level, for example a decrease by at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% decrease(e.g. absent level as compared to a reference sample), or any decreasebetween 10-100% as compared to a reference level.

The terms “increased,” “increase” or “enhance” or “activate” are allused herein to generally mean an increase by a statically significantamount; for the avoidance of any doubt, the terms “increased”,“increase” or “enhance” or “activate” means an increase of at least 10%as compared to a reference level, for example an increase of at leastabout 20%, or at least about 30%, or at least about 40%, or at leastabout 50%, or at least about 60%, or at least about 70%, or at leastabout 80%, or at least about 90% or up to and including a 100% increaseor any increase between 10-100% as compared to a reference level, or atleast about a 2-fold, or at least about a 3-fold, or at least about a4-fold, or at least about a 5-fold or at least about a 10-fold increase,or any increase between 2-fold and 10-fold or greater as compared to areference level.

The term “statistically significant” or “significantly” refers tostatistical significance and generally means at least two standarddeviations (2SD) away from a reference level. The term refers tostatistical evidence that there is a difference. It is defined as theprobability of making a decision to reject the null hypothesis when thenull hypothesis is actually true.

The term “modulator” refers to a compound that alters or elicits anactivity of a molecule. For example, a modulator may cause an increaseor decrease in the magnitude of a certain activity of a moleculecompared to the magnitude of the activity in the absence of themodulator. In certain embodiments, a modulator is an inhibitor, whichdecreases the magnitude of one or more activities of a molecule. Incertain embodiments, an inhibitor completely prevents one or moreactivities of a molecule. In certain embodiments, a modulator is anactivator, which increases the magnitude of at least one activity of amolecule. In certain embodiments the presence of a modulator results inan activity that does not occur in the absence of the modulator. Withoutlimitations, a modulator can be selected from the group consisting ofsmall or large organic or inorganic molecules; monosaccharides;disaccharides; trisaccharides; oligosaccharides; polysaccharides;biological macromolecules, e.g., proteins, peptides, peptide analogs andderivatives thereof, peptidomimetics, nucleic acids, nucleic acidanalogs and derivatives, enzymes, antibodies, portion or fragments ofantibodies; an extract made from biological materials such as bacteria,plants, fungi, or animal cells or tissues; naturally occurring orsynthetic compositions; and any combinations thereof.

The term “selective modulator” refers to a compound that selectivelymodulates a target activity.

The term “selectively modulates” refers to the ability of a selectivemodulator to modulate a target activity to a greater extent than itmodulates a non-target activity.

The term “target activity” refers to a biological activity capable ofbeing modulated by a modulator. Certain exemplary target activitiesinclude, but are not limited to, binding affinity, signal transduction,enzymatic activity, and the like.

The term “agonist” refers to a compound, the presence of which resultsin a biological activity of a receptor that is the same as thebiological activity resulting from the presence of a naturally occurringligand for the receptor.

The term “partial agonist” refers to a compound the presence of whichresults in a biological activity of a receptor that is of the same typeas that resulting from the presence of a naturally occurring ligand forthe receptor, but of a lower magnitude.

The term “antagonist” refers to a compound, the presence of whichresults in a decrease in the magnitude of a biological activity of areceptor. In certain embodiments, the presence of an antagonist resultsin complete inhibition of a biological activity of a receptor.

The term “inhibitor” refers to molecules or substances or compounds orcompositions or agents or any combination which are capable ofinhibiting and/or reducing the activity of the target molecule. As usedherein, the term “inhibitor” is interchangeable with the term“antagonist”. The term “inhibitor” comprises competitive,non-competitive, functional and chemical antagonists. The term “partialinhibitor” means a molecule or substance or compound or composition oragent or any combination thereof that is capable of incompletelyblocking the action of agonists through, inter alia, a non-competitivemechanism.

As used herein, the term “ligand” refers to an agent that binds to atarget molecule. A ligand is not limited to an agent that binds to arecognized functional region of the target molecule, e.g., the activesite of an enzyme, the antigen-combining site of an antibody, thehormone-binding site of a receptor, a cofactor-binding site, and thelike. A ligand can also be an agent that binds any surface orconformational domains of the target compound. Therefore, the ligandsencompass agents that in and of themselves may have no apparent or knownbiological function, beyond their ability to bind to the target in themanner described above. The term ligand encompasses agents that reactupon binding and agents that do not react other than by binding.

As used herein, the term “small molecule” can refer to compounds thatare “natural product-like,” however, the term “small molecule” is notlimited to “natural product-like” compounds. Rather, a small molecule istypically characterized in that it contains several carbon-carbon bonds,and has a molecular weight of less than 5000 Daltons (5 kD), preferablyless than 3 kD, still more preferably less than 2 kD, and mostpreferably less than 1 kD. In some cases it is preferred that a smallmolecule have a molecular weight equal to or less than 700 Daltons.

The disclosure is further illustrated by the following examples whichshould not be construed as limiting. The examples are illustrative only,and are not intended to limit, in any manner, any of the aspectsdescribed herein. The following examples do not in any way limit theinvention.

EXAMPLES Example 1—Screening Assay

Satellite cells were harvested from CAG-B-actin-GFP mice, aged 2-4months old via the FACS isolation method outlined in Sherwood et al 2004and Cerletti et al 2008. Briefly, all limb muscles and abdominal muscleswere harvested from the animals and digested first in a 0.2% collagenasesolution, then in a 0.0125% collagenase/0.05% dispase solution. Thefiltered solution was stained for cell surface markers, and put throughFACS. Cells that were negative for Mac1, Sca1 and CD45 and positive forBeta-integrin 1 and CXCR4 were used for the screening assay. Cells wereplated directly from the FACS machine at 50 cells/well, into 96-wellplates coated with 10 ug/mL laminin (4-6 hours at 37 C, then partiallyremoved). Media for the screen was Ham's F-10 supplemented with 10%heat-inactivated horse serum, 1× penicillin/streptomycin and 1×L-glutamine. Basic FGF (bFGF) was added only to the positive controlwells; all other wells received media only. The day of plating wascalled Day 0. The day after plating, Day 1, compounds were added. Allcompounds were dissolved in DMSO, and initially tested at 10 uM, 1 uM or0.1 uM in duplicate. The negative control for each plate was the sameconcentration of DMSO (no compounds dissolved). Compounds were incubatedwith the cells until Day 4, with no media change. bFGF was spiked intopositive control wells daily. On Day 4, plates were fixed in 4%paraformaldehyde and washed with phosphate buffered saline. Plates wereimaged directly on the Opera Confocal imager, since the cells werereadily visible from the CAG-EGFP-Beta-actin transgene. An Acapellascript was used to count the number of cells in each well. Wells werescored for proliferation based on the cell counts (DMSO-treated wellsusually had around 50 cells at the end of the assay, bFGF-treated wellsusually had 150-250 cells at the end). We selected compounds as hits ifthey had cell numbers greater than or equal to one (or, for the secondsession of screening, three) standard deviation(s) from the DMSOcontrols. Compounds thus selected were then tested in an initial dosecurve, usually concentrations between 20 nM-30 uM commensurate with theconcentration flagged as a hit. We called compounds active if they wereable to cause proliferation to cell numbers at least two standarddeviations over the DMSO negative control. Any compounds that were foundto be active in the dose curve were re-supplied from the originalvendors. Re-supplied compounds were again tested in a dose curve forproliferation ability. Compounds still found to be active were then putin the queue for optimization and characterization.

TABLE 1 Compounds Screened in Session One Composition by Target ClassNo. of compounds Kinase Inhibitors 106 GPCR Drug List 20 Diverse set ofGPCR Ligands 78 Others 11 Total 215

TABLE 2 Compounds Screened in Session Two Composition by Target ClassNo. of compounds HDAC 10 Hh Ag + An 17 Neuropeptides 5 GPCR-Dopamine 29GPCR-Serotonin 31 GPCR-Histamine 27 g-secretase (notch) 7 ionophore 5Ion channels 25 Kinase Inhibitors 39 Total 195

As described above, the inventors screened a set of 215 compoundscomprised primarily of kinase inhibitors and GPCR ligands. The inventorsassayed these compounds at 1 μM, and counted as potential hits thosecompounds that scored greater than one standard deviation over theDMSO-treated negative control. Using these criteria, the inventorsidentified 20 compounds from the set as hits. The inventors then testedeach of these 20 compounds in a dose response assay to validate theiractivity. Five compounds found to have activity using this method areshown in Table 3. Dose response curves for the five compounds are shownin FIGS. 9A-13.

TABLE 3 Library Initial Compound Source hit Conc. Target LestaurtinibKinases 1 uM Flt3 Kinase, (CEP-701) Jak2, ROR gamma? SU 11652 Kinases 1uM PDGFR/EGFR SU 11248 Kinases 1 uM EGFR, Flt3 (Sunitinib) kinaseBosutinib Kinases 1 uM Bcr-abl, SRC (SKI 606) kinase JAK3 Kinases 1 uMInhibitor VI

Three of the compounds (Sunitinib (SU11248), JAK3 inhibitor VI, andLestaurtinib/CEP701) were retested and found to be active in the retest.Each compound promoted proliferation of the input SMP cells similar tothat seen in the bFGF positive control (4.7±1.4 for Sunitinib andLestaurtinib, 3.7±1.1 for JAK3 inhibitor VI). The DMSO-treated cellsproliferated to a level of 1±0.3 in both trials.

In addition, the inventors also examined whether CEP 701 can synergizewith bFGF to achieve even higher levels of SMP proliferation. For theseexperiments, 300 cells were plated in each well instead of the 50cells/well used for the screen and subsequent dose response assays. Thischange in protocol was introduced in an effort to reduce the variabilityin the assays. Under these conditions 0.05 μM CEP701 was added the dayafter plating the purified SMPs, and 5 ng/mL bFGF was added to theappropriate samples on a daily basis. Three days after plating the mediawas changed and fresh compound was added. This additional change in theprotocol was introduced both to improve viability and reducevariability. The plates were fixed four days after plating.

Cultures treated with both CEP701 and bFGF showed an 8-fold increase inproliferation (FIG. 14), indicating that CEP701 and bFGF can actadditively to increase SMP expansion in vitro. Because changing themedia seemed to lead to increased viability in the cells, the inventorsdecided to try removing the compounds several days before fixation toallow the cells to recover. For the following experiments, compoundswere added the day after plating (Day 1). Compound was refreshed daily,until it was removed from the cultures completely on the day asindicated below. Cells were then cultured in media without supplementedcompounds until fixation on Day 4. The inventors discovered that twodays of recovery time was advantageous to the cultures. The inventorsconfirmed that discovered that CEP-701 can work additively with bFGF(FIG. 15). Additionally, while Jak3 inhibitor VI also worked additivelywith bFGF (FIG. 16), Sunitinib did not worked additively with bFGF (FIG.17).

In order to assess the effects these compounds have on the identity ofthe SMPs, the inventors studied the ability of the treated SMPs todifferentiate in culture. For these experiments, 300 cells were platedin each well at Day 0 in our standard proliferation media (10% horseserum in F-10). Compounds were added at Day 1 and SMPs were allowed toproliferate for three days. At Day 4, the media was switched todifferentiation media (10% horse serum, 10% FBS, 0.5% chick embryoextract in high-glucose DMEM). Cultures were then incubated for three orfour days under differentiation conditions and fixed on Day 7 or Day 8.They were then stained with Hoechst and anti-myosin heavy chainantibody.

As shown in FIGS. 18-20, treatment with CEP701, Sunitinib, or Jak3inhibitor VI did not affect the ability of expanded SMPs todifferentiate and fuse to form myotubes. Additionally, satellite cellsthat had been exposed to CEP701 for 3 days and then allowed to recoverfor two days before being put into differentiation conditions were alsoable to fuse into myotubes (data not shown).

The inventors focused on CEP701 for further studies. The inventors nextstudied additive effect of CEP701 with TGF-beta inhibitors. TGF-Betainhibitors are known to promote proliferation and preventdifferentiation of satellite cells. Addition of Alk5 inhibitor II to thecultures produced a slight increase in proliferation. However, littleadditive effect was seen on addition of CEP701 and TGF-beta inhibitor(FIG. 21).

Next the inventors tested the specificity of CEP701 for proliferatingsatellite cells. The inventor isolated the Sca1-positive population offibroblasts from the muscle preparation, obtained via FACS. Either 500or 3000 fibroblasts were plated in each well, and cultured in DMEMsupplemented with 10% FBS. Compound was added the day after plating, andrefreshed daily. Plates were fixed five days after plating and stainedfor the proliferation marker Ki67. As seen in FIG. 22, no difference inthe percentage of proliferating cells between those exposed to CEP701(right panel) and those exposed to DMSO (left panel) was seen. Inaddition, CEP701 had no effect on primary fibroblasts (FIG. 23).

In another experiment, the inventors tested the CEP701 compound in agedtissue. The aged mice were 15 months old at the time of the experiment.The conditions used for the assay were the same as those used for younganimals. After FACS, 300 cells/well were plated on Day 0 in theinventors' standard proliferation media (10% horse serum in F-10).Compound was added at Day 1 at indicated concentrations, and refresheddaily. At Day 4, compound was withdrawn and replaced with media only.Plates were fixed and imaged at Day 6.

As seen in FIGS. 24 and 25, 50 nM CEP701 appeared to be the optimumconcentration for aged cells as well as young cells. In both cases,exposure to 50 nM CEP701 under the described conditions resulted inapproximately a six-fold increase in the number of cells. CEP701 andbFGF were able to act additively in cultures of old cells, producingapproximately a ten-fold increase in cell number.

In another experiment, the inventors screened a new set of 200compounds. This set was primarily focused on GPCR ligands, with somekinase inhibitors and other annotated compounds. The inventors screenedthe compounds at 10 uM in duplicate, except the kinase inhibitors whichwere screened at 1 uM in duplicate. Compounds were scored as potentialhits if they increased proliferation of input SMPs at levels greaterthan three standard deviations over the negative control in either ofthe replicates. Using these criteria, the inventors identified andvalidated 2 hits (N6-cyclopentyladenosine, an adenosine receptoragonist, and Budesonide, a glucocorticoid steroid) in subsequent doseresponse curves (FIGS. 26 and 27).

After additional testing, we ultimately decided to resupply Budesonide,and N6-cyclopentyladenosine (CPA) from the original vendor in order totest a different batch of the compounds. We performed dose responses onall the re-supplied compounds, plating 50 cells in each well as we haddone during the screen and the first dose response tests

In this experiment, the DMSO negative control had a normalized value of1±0.4 and the bFGF positive control had a value of 5.6±1.7. Both CPA andBudesonide were effective and produced proliferation levels much greaterthan that seen in the negative control. Without wishing to be bound by atheory, repeating these assays under the optimized experimentalconditions the inventors have developed (300 cells/well plus a mediachange to refresh compounds after two days exposure) can improve boththe variability of the assay as well as the proliferation response.

As with the hits from the first set of compounds screened, the inventorstested these compounds for ability to synergize with bFGF. Usinginventors' standard culture conditions (10% horse serum in F-10),compounds were added to cells the day after plating, and refreshed dailyuntil removed from the cultures as indicated. Cells were then grown inmedia without supplemented compounds until fixation on Day 4.

CPA (30 μM) was seen to synergize with bFGF in vitro. The combination ofCPA and bFGF showed a fifteen-fold increase in the cell number, ascompared with approximately a six-fold increase with bFGF alone (FIG.28). However, combination of Budesonide with bFGF provided no additionalincrease as compared to bFGF alone (FIG. 29).

The inventors also assessed effects these compounds were having on theidentity of the satellite cells and their ability to differentiate inculture. For these experiments, 300 cells were plated in each well atDay 0 in our standard proliferation media (10% horse serum in F-10).Compounds were added at Day 1 and SMPs were allowed to proliferate forthree days. At Day 4, the media was switched to differentiation media(10% horse serum, 10% FBS, 0.5% chick embryo extract in high-glucoseDMEM). Cultures were then incubated for three or four days underdifferentiation conditions and fixed on Day 7. They were then stainedwith Hoechst and anti-myosin heavy chain antibody. As can be seen in theFIGS. 29 and 30, satellite cells exposed to the compound are able tofuse into myosin heavy chain-positive myotubes.

The inventors then tested whether these compounds can work additivelywith TGF-Beta inhibitors. A slight increase in proliferation was seenwhen the cells were treated with both CPA and Alk5 inhibitor II ascompared to CPA and Alk5 inhibitor II alone (FIG. 31).

In this study, the inventors carried out a screen designed to identifycompounds that are able to cause satellite cells to proliferate invitro. The inventors discovered several compounds that can causeproliferation, both in the presence and absence of bFGF. The compoundsgave rise to satellite cell populations that differentiate normally andcarried normal markers for differentiation state. These results showthat treatment with these compounds allow the satellite cell populationto proliferate normally and contribute to muscle repair in diseasestates.

Example 2

Satellite cells were harvested from CAG-B-actin-GFP mice, aged 2-4months old via the FACS isolation method as outlined in Example 1.Briefly, all limb muscles and abdominal muscles were harvested from theanimals and digested first in a 0.2% collagenase solution, then in a0.0125% collagenase/0.05% dispase solution. The filtered solution wasstained for cell surface markers, and put through FACS. Cells that werenegative for Mac1, Seal and CD45 and positive for Beta-integrin 1 andCXCR4 were used for the screening assay. Cells were plated directly fromthe FACS machine at 50 cells/well, into 96-well plates coated with 10ug/mL laminin (4-6 hours at 37° C., then partially removed). Media forthe screen was Ham's F-10 supplemented with 10% heat-inactivated horseserum, IX penicillin/streptomycin and IX L-glutamine. Basic FGF (bFGF)was added only to the positive control wells; all other wells receivedmedia only. The day of plating was called Day 0.

The day after plating (Day 1) the compounds were added, as illustratedin FIG. 33. All compounds were dissolved in DMSO, and initially testedat 10 uM, 1 uM or 0.1 uM in duplicate. The negative control for eachplate was the same concentration of DMSO (no compounds dissolved).Compounds were incubated with the cells until Day 4, with no mediachange. bFGF was spiked into positive control wells daily. On Day 4,plates were fixed in 4% paraformaldehyde and washed with phosphatebuffered saline. Plates were imaged directly on the Opera Confocalimager, since the cells were readily visible from theCAG-EGFP-Beta-actin transgene. An Acapella script was used to count thenumber of cells in each well. Wells were scored for proliferation basedon the cell counts (DMSO-treated wells usually had around 50 cells atthe end of the assay, bFGF-treated wells usually had 150-250 cells atthe end), Compounds were selected as hits if they had cell numbersgreater than or equal to one (or, for the second session of screening,three) standard deviation(s) from the DMSO controls. Compounds thusselected were then tested in an initial dose curve; usuallyconcentrations between 20 nM-30 uM commensurate with the concentrationflagged as a hit. Compounds were deemed to be active if they were ableto cause proliferation to cell numbers at least two standard deviationsover the DMSO negative control. Any compounds that were found to beactive in the dose curve were re-supplied from the original vendors.Re-supplied compounds were again tested in a dose curve forproliferation ability. Compounds still found to be active were then putin the queue for optimization and characterization.

The inventors screened a set of approximately 400 compounds from thecustom screening library illustrated in FIG. 34. The inventors assayedthese compounds at 1 μM, and counted as potential hits those compoundsthat scored greater than one standard deviation over the DMSO-treatednegative control. Using these criteria, the inventors identifiedapproximately 10 compounds from the set as hits that were capable ofincreasing in vitro satellite cell proliferation. The inventors thentested each of these compounds in a dose response assay to validatetheir activity. As illustrated in FIG. 35, four of the compounds thatwere found to increase in vitro satellite cell proliferation wereLestaurtinib (CEP701), Sunitinib (SU11248), JAK3 inhibitor VI, andN6-cyclopentyladenosine (CPA). Lestaurtinib (CEP701) was identified as atop hit, was effective at nanomolar doses and had target overlap withseveral other hit compounds. Additionally, Lestaurtinib (CEP701)increased proliferation of aged satellite cells in vitro (FIG. 36),increased proliferation of human satellite cells and did not increaseproliferation of fibroblasts.

The inventors then tested each of these compounds in a dose responseassay to validate their activity, as depicted in FIG. 37A. Dose responsecurves for Lestaurtinib (CEP701), Sunitinib (SU11248), JAK3 inhibitorVI, and N6-cyclopentyladenosine (CPA) are shown in FIG. 37B.

In addition to Lestaurtinib (CEP701), Quizartinib (AC220), a smallmolecule receptor tyrosine kinase inhibitor, also demonstrated anability to expand human satellite cells at low doses. As illustrated inFIG. 38, both CEP-701 and AC220 increased human satellite cells by morethan 2-fold at a concentration of 1 nM relative to the DMSO control.

As illustrated in FIG. 39, a differentiation media comprising 5% horseserum and those compounds identified as hits (e.g., CEP701, SU11248,JAK3 inhibitor VI, CPA and Tyr AG490) drive myoblast differentiation.Similarly, FIG. 40 demonstrates that CEP701 enhances myoblastdifferentiation in differentiation media relative to the DMSO control,as evidenced by the observed increase in both myoblast area and length.

The present inventors next sought to determine whether CEP701-treatedcells retain engraftability by performing the experimental protocoldepicted in FIG. 41A, by engrafting tubulin>GFP satellite cells intoinjured mdx muscle and administering CEP701 or a DMSO control. Asillustrated in FIG. 41B, CEP701 treated cells resulting in an increasednumber of GFP+ fibers per section, relative to the DMSO control.

The present inventors next sought to determine whether CEP701 treatmentincreases regenerating fiber size and satellite cell number in vivo.CEP701 was administered subcutaneously to the mice post-CTX injury,followed by harvesting of the tissue, as depicted in FIG. 42A.Regenerating muscle fibers are eMHC+. As illustrated in FIGS. 42B-42D,treatment with CEP701 increased both regenerating fiber size andsatellite cell number in vivo in both adult and aged mice.

The present inventors also performed an in vitro binding assay againstactive site fragments (KINOMEscan) in an effort to identify thosemultiple hit compounds that inhibit receptor tyrosine kinases (RTKs). Asshown in Table 4 below, CEP701, AC220 and sunitinib each inhibitmultiple RTKs (numbers are Kd in nM; expression in primary myoblasts).

TABLE 4 CEP- qPCR* Kinase Ligand 701 AC220 Sunitinib FACS ImagingLiterature (Ct) Flt3 FL 8.5 1.6 0.47 NO ~27-28 Flt4 VEGF 17 41 50 ~32-33RET GDNF 20 9.9 12 YES YES ~27-28 PDGFRβ PDGF 28 7.7 0.075 NO ~27-28 M-CSF1 110 12 2 NO ~30-34 CSF1R VEGFR2 VEGF 110 87 1.2 ~31-33 FLT1 VEGF120 41 1.8 Yes? ~31-32 c-KIT SCF 150 4.8 0.37 ~33 PDGFRα PDGF 380 110.79 NO ~31-32 DDR1 Collagen 510 81 2000

RET proto-oncogene is the receptor for the GDNF ligand family, whichincludes glial derived neurotrophic factor, neurturin, artemin andpersephin, and is important for the development and function of severaltissue types, including the nervous system. The RET proto-oncogeneactivates several downstream pathways, including JAK/STAT. Asillustrated in FIG. 43A, the multiple hit compounds CEP701, AC220 andsunitinib that are identified in Table 4 inhibit the PDGFR family ofRTKs. Phospho-RET levels are elevated in injured muscle, as illustratedin FIG. 43B, which compares the fold change in phospho-RET in bothuninjured contralateral tibialis anterior (TA) muscle to that observed 2days post-cardiotoxin injury. As illustrated in FIG. 43B, 2 dayspost-cardiotoxin injury, an approximately 8-fold increase in phospho-RETwas observed by ELISA relative to uninjured contralateral TA muscle.

Satellite cells express RET in vitro, as shown in FIGS. 44A-44B. Asillustrated in FIGS. 45A-45D, CEP701 treatment inhibits RETphosphorylation in vitro.

FIG. 46 shows the results of a study evaluating the effects of the invitro deletion of RET. Using a conditional RET mutant and reporter, thepresent inventors were able to determine that the RET promoter is activein at least 25% of satellite cells (FIG. 47) and that RET knockout cellsproliferate better than wild-type cells in vitro (FIG. 48). FIG. 49demonstrates the fold change relative to control of untreated FLT3 andRET knockout cells.

All patents and other publications identified in the specification andexamples are expressly incorporated herein by reference for allpurposes. These publications are provided solely for their disclosureprior to the filing date of the present application. Nothing in thisregard should be construed as an admission that the inventors are notentitled to antedate such disclosure by virtue of prior invention or forany other reason. All statements as to the date or representation as tothe contents of these documents is based on the information available tothe applicants and does not constitute any admission as to thecorrectness of the dates or contents of these documents.

Although preferred embodiments have been depicted and described indetail herein, it will be apparent to those skilled in the relevant artthat various modifications, additions, substitutions, and the like canbe made without departing from the spirit of the invention and these aretherefore considered to be within the scope of the invention as definedin the claims which follow. Further, to the extent not alreadyindicated, it will be understood by those of ordinary skill in the artthat any one of the various embodiments herein described and illustratedcan be further modified to incorporate features shown in any of theother embodiments disclosed herein.

What is claimed is:
 1. A method of increasing satellite cellproliferation, the method comprising: contacting a satellite cell withvorinostat, in an amount effective to increase satellite cellproliferation.
 2. The method of claim 1, wherein the contacting is invivo.
 3. The method of claim 2, wherein the in vivo contacting is in ahuman.
 4. The method of claim 2, wherein the in vivo contacting is in asubject, wherein the subject is in need of treatment for damaged muscletissue.
 5. The method of claim 4, wherein the damaged muscle tissue isthe result of a physical injury or accident, disease, infection,over-use, loss of blood circulation, or muscle atrophy or wasting. 6.The method of claim 4, wherein the damaged muscle tissue is the resultof muscle atrophy/wasting.
 7. A method for muscle repair or regenerationin a subject having damaged muscle tissue, the method comprisingadministering to the subject vorinostat, in an amount effective toincrease satellite cell proliferation.
 8. The method of claim 7, whereinthe subject is human.
 9. The method of claim 7, wherein the damagedmuscle tissue is the result of a physical injury or accident, disease,infection, over-use, loss of blood circulation, or muscle atrophy orwasting.
 10. The method of claim 7, wherein the damaged muscle tissue isthe result of muscle atrophy/wasting.